Structural Biochemistry/Volume 2

Molecular Organization edit

The Cell and Its Organelles edit

The cell is the most fundamental unit of living organisms, providing both structure and function. Different cells may take on different shapes, sizes, and functions, but all have the same fundamental properties. Within the cell are various organelles, which give the cell structure and function. The amounts and types of organelles found vary from cell to cell.[1]

There are two major types of cells: prokaryotes and eukaryotes. A prokaryotic cell, such as a bacteria cell, is one which lacks a "true" nucleus and membrane-bound organelles. The genetic information of a prokaryote is localized in the nucleoid region within the cytoplasm. On the other hand, eukaryotic cells store their genetic information in a membrane-enclosed nucleus. The larger of the two types, eukaryotes further differ from prokaryotes in that they contain membranous organelles, such as the mitochondria and the endoplasmic reticulum.

Two common types of eukaryotic cells are animal and plant cells. Plant cells have cell walls, which give them their rigid structure. Unique to plant cells, chloroplasts allows them to photosynthesize. These two structures are not found in animal cells. The absence of a cell wall enables animals cells to adopt a variety of shapes. A lysosome, which contains digestive enzymes, is found exclusively in animal cells. Another difference between animal and plant cells is the size of their vacuoles. Animal cells have several small vacuoles throughout the cytoplasm while plant cells contain just one large central vacuole.

Cell Size edit

The size of a cell is limited by diffusion. Though larger cells are able to contain more biochemicals, a smaller size that maximizes the surface area-to-volume ratio is most ideal. Simple geometry tells us that the area-to-volume ratio is given by 3/r, which is inversely proportional to the radius. Molecules, such as oxygen, that are required by the cell to function need to be able to reach all parts of the cell efficiently. Thus, a greater diffusion rate is promoted by a smaller cell size.[2]

Cell Movement edit

One of the most traveled cells are blood cells. Red blood cells move passively as the bloodstream carries them. White blood cells protect people from viruses and bacteria so their movement needs to be quick to the site of infection. The feeling of pain is what happens when white blood cells move to the focused site. The white blood cells move like amoebae, where they move by stretching parts of themselves and the entire cell moves towards where said part was stretched. This process is repeated over and over again. [3]

Cell Theory edit

The Cell Theory is that all living things are composed of cells and that all cells originate from pre-existing cells. Cells are relatively small because it reassures a big surface to volume ratio, which is need to facilitate a fast exchange of material with the extracellular environment.

Supramolecular Complexes edit

Supramolecular complexes compose the organelles. These include chromosomes, plasma membrane, and the cell wall.

Macromolecules edit

Supramolecular complexes are composed of macromolecules, such as DNA, protein, and cellulose. Macromolecules are the rather large in size molecules in chemistry. The weak, non-covalent forces between macromolecules provide stability and structure to the supramolecular complex. The four major macromolecules are nucleic acids, proteins, sugars, and lipids. Macromolecules needs assistance, such as salts or ions, when dissolving. In the case of proteins, they will denature when the concentration is out of their range of concentration. Since they are so big, they will also effect the rate of equilibrium when there is a very high concentration of macromolecules.


Monomeric Units edit

Macromolecules can be further subdivided into their respective monomeric units. DNA is composed of nucleotide units, while protein is composed of amino acids. Carbohydrates are made of sugar subunits, and lipids are made of fatty acid subunits.

The Structural Hierarchy edit

Supramolecular complex Macromolecules Monomeric Units
Chromosome DNA Nucleotides
Plasma membrane Protein Amino Acid
Cell Wall Cellulose Sugars

References edit

  1. Nelson, David L. (2004). Principles of Biochemistry (4th Ed. ed.). W. H. Freeman. ISBN 0716743396. {{cite book}}: |edition= has extra text (help)
  2. Nelson, David L. (2004). Principles of Biochemistry (4th Ed. ed.). W. H. Freeman. ISBN 0716743396. {{cite book}}: |edition= has extra text (help)
  3. Inside the Cell
  4. Nelson, David L. (2004). Principles of Biochemistry (4th Ed. ed.). W. H. Freeman. ISBN 0716743396. {{cite book}}: |edition= has extra text (help)
Pictorial representation of organelles in a typical animal cell

Structural biochemistry plays a vital role in the functions of an organism's cell through various means, one of them being the organelles in a cell. It is through the structure and functions of living molecules (and some non-living), such as nucleic acids, amino acids, purine, and lipids that life is even possible.

Some properties of living organisms include high degree of chemical complexity and microscopic organization, systems to extract, transform and use energy from the environment, self-replication and self-assembly, sensing and responding to changes in the environment, define functions for each component and regulation among them, and history of evolutionary change.

Organelles are the components of the cell that synthesize new materials, recycle old materials, transport molecules, and anything else that is essential to ensure the proper survival of the cell and its propagation. Organelles incorporate all broad ranges of organic molecules including nucleic acids, amino acids, carbohydrates, and lipids to produce a viable cell.

As discussed before, the "lipid bilayer" that forms the cell membrane contains membrane protein and cholesterol. The membrane protein plays a vital role in the membrane functions while the cholesterol performs the structural role within the membrane.

There are two types of protein membranes:

  • Integral membrane protein: lies within the membrane
  • Peripheral membrane protein: bound to membrane

The cell membrane is often referred to as a mosaic. Proteins in the membrane determine most of the membranes specific function. These proteins are categorized as integral and peripheral proteins. Integral proteins can perform a number of functions such as being transport proteins that provide a hydrophilic channel across the membrane. Integral proteins can also act a receptor sites for chemical messengers like hormones. Enzymes can also be found in the lipid bilayer with its active site exposed to substances in adjacent solutions. Elements of the cytoskeleton may also be bonded to the membrane proteins; a function that helps maintain cell structure. The structure of these membrane protein can also be either alpha helices or beta sheets. Due to the hydrophobic interactions, the hydrophobic residues of the alpha helices will not exposed to the aqueous environment. The beta sheet forms a hollow cylindrical configuration where the inside is hydrophilic. The cylindrical structure is driven by the unsatisfied hydrogen bonding at the ends of the beta sheet. Wrapping around itself, the beta sheet is able to satisfy all hydrogen bonding.

The major functions performed by proteins of the plasma membrane are:

  1. Transport: Some membrane proteins provide selective hydrophilic channels for exchange of substances.
  2. Enzymatic activity: Membrane proteins may also be enzymes with their active sites exposed to the surrounding solutions.
  3. Signal transduction: Membrane proteins may act as receptors with specific binding sites that allows perfect fit with chemical messengers, which can cause the protein to change shape and allow it to relay the message to the inside of the cell.
  4. Cell-cell recognition: Some glycoproteins allow specific identification by membrane protein of other cells.
  5. Intercellular joining: Membrane proteins of adjacent cells may join together in different junctions.
  6. Attachment to the cytoskeleton and extracellular matrix (ECM): Membrane proteins can be non-covalently bound with elements of the cytoskeleton in order to maintain cell shape and stabilize the location of certain membrane proteins.

Cholesterol regulates the fluidity of the membrane in eukaryotic cells. The ability to incorporate cholesterol into the cell membrane with hydrophobic and hydrophilic interactions allows the cholesterol to disrupt the phospholipid interaction within the bi-layer. Since prokaryotic cells do not have cholesterol to regulate fluidity, these cells depend on the variation in the saturation level and length of the fatty acid chain. The shorter and more saturated the chain the more rigid the membrane will become (due to the fact that the longer and saturated chains can interact more closely with one another)

Fluid Mosaic Model edit

This model claims that the lipid layer has an important role in cell membrane. The cell membrane serves as the solvent for the integral membrane proteins, and it also serves as a barrier that separates the cellular activities within the cell from the extracellular space. The permeable barrier regulates what enters the cell. This Fluid Mosaic Model is regulated by the concentration of cholesterol and fatty acid chain mentioned above.

The Properties of Membrane edit

  • Sheet-like structure
  • Formed by lipid bilayers and proteins
  • The different ratio of lipids to proteins will correspond to the different cell types and organelles which gives it the amphiphatic properties
  • Non-covalent assemblies include van deer Waals, hydrogen bonding, and hydrophobic interactions
  • Asymmetric
  • The orientation of the proteins are fixed and will not interchange between the inner or outer layers
  • Fluid Structures
  • Electrically polarized because of the charged head groups

Major Organelles edit

1. Ribosome edit

Ribosomes are the sites by which nucleic acids are translated and proteins are synthesized. Ribosomes are about 20 nm in diameter and are composed of ribosomal RNA and proteins. They can be found freely floating in the cytosol and not attached to any organelles in prokaryote cells. In eukaryote, ribosomes may be found on the rough endoplasmic reticulum. The rough ER earns its name because of ribosomes on its surface, giving a studding appearance. The proteins produced by the ribosomes of the rough ER are sent through the lumen of the ER, where they are modified. The protein is then transported in a vesicle to the Golgi Apparatus, where the protein undergoes further modification.

First, the genetic code from DNA is transcribed into a complementary strand called messenger RNA (mRNA) (mRNA) by DNA polymerase. In prokaryotes, the mRNA moves away from the nucleoid and is bounded to free-floating ribosomes in the cytosol. However, in eukaryotes, mRNA is made in the nucleus and transported across the nuclear membrane and into the cytoplasm. This is called translocation. In the next step, known as translation, the mRNA is attached to the ribosome, and codons on the mRNA are matched with the complementary nucleotide bases (anticodons) located on a transfer RNA (tRNA) molecule. The enzyme aminoacyl tRNA synthetase matches the tRNA codons with the appropriate amino acids through a series of esterification reactions. Ribosomal RNA synthesizes the protein through use of RNA polymerase. This elongates the protein until a stop codon terminates the protein synthesis chain. The synthesis of proteins always moves in the direction of the N-terminus to the C-terminus. DNA replication also follows the 5' to 3' direction.

2. Cell Membrane edit

The cell membrane, as explained above, is a selectively permeable barrier of ions and molecules that move into and out of the cell. In other words, not all molecules are able to pass through the cell membrane. During the division of the cell, none of the membrane integrity is lost. As the cell grows, new lipid and protein molecules are placed into the cell’s plasma membrane.

Prokaryotes Organelles edit

Prokaryotes typically have no compartmentalized organelles. The cell's DNA and ribosomes are free-floating with the cytosol, which is surrounded by a cell membrane. A prokaryotic cell is generally one hundred times smaller than a eukaryotic cell.

1. Nucleoid edit

In the nucleoid, the chromosomal DNA is wrapped around binding proteins. "Replication by DNA polymerase and transcription by RNA polymerase occur at the same time within the nucleoid." [1]

2. Pili edit

Pili (Fimbriae)is a thin structure that stick out from the surface. They are made out of a single protein called pilin. Pili's functions include DNA transfer, binding to surfaces, and motility. Pili has the ability to attach to a substrate. One type of pili called sex pili, it attaches a "male" donor cell to a "female" recipient cell for transfer of DNA. This process is called conjugation.

Eukaryotes Organelles edit

Eukaryotes include animals, plants, fungi, and protists. They are typically more complex than prokaryotic cells. There are many compartmentalized organelles enclosed in membranes within the cell which allow for various reactions to take place. The eukaryotic cell is typically much larger than the prokaryotic cell, usually by a factor of around 100. Each organelle in Eukaryotes has their own function in the cell.

1. Nucleus edit

The nucleus is one of the primary organelles that distinguish eukaryotic cells from prokaryotic cells. It is an organelle that enclosed compartment with a specific function. It contains chromatin. Nuclei contains nucleolus (where ribosome assembly is). Ribosomal RNA get together with ribosomal proteins to form the ribosomal subunits. The nucleus contains DNA that house the genes coding for the synthesis of proteins, antibodies and molecules that perform the basic functions of the cell. The nuclear membrane contains nuclear pore conplexes that allow for transport of material into and out of membrane. They also export mRNAs out of the nucleus. [2]

2. Endoplasmic Reticulum edit

There are two types of endoplasmic reticulum: smooth ER and rough ER. The smooth ER is the site of lipid synthesis and some detoxification of noxious compounds. The rough ER is the site where transmembrane proteins or secreted proteins are translocated. Ribosomes are located in the rough ER instead of smooth ER because the protein has a hydrophobic signal sequence on its amino terminus. Endoplasmic Reticulum is where proteins can be modified too.

3. Mitochondrion edit

Mitochondria (Singular: Mitochondrion) involved in cellular energy production. It has a function in performing oxidative respiration and are found in nearly all eukaryotes. Mitochondria also produces ATP by oxidative respiration. It also has an outer and an inner membrane. Both DNA and ribosomes of mitochondria show similarities with DNA and ribosomes of bacteria.

4. Golgi Apparatus edit

Proteins that are not part of ER now move to the Golgi. Golgi complex has membrane stacks (cisternae) that each contain unique enzymes. Carbohydrates may be modified as proteins pass through the cisternae. Vesicles leaving the Golgi complex may fuse with the cell membrane.

5. Centriole edit

Centrioles are involved in a process called nuclear division. They are small, self-replicating, and are located in the cytoplasm near the nucleus. organelles present near nucleus of animal cells.

6. Cell Wall edit

The cell wall, located outside the cell membrane, is a tough layer that provides the rest of the cell with structure support and protection. Cell walls are present in plants, fungi, algae, some archaea, and bacteria cells, but not in animal cells. The cell wall confers the shape and rigidity to bacterial cell and helps it withstand the intracellular turgor pressure that can build up as a result of osmotic pressure.

7. Chloroplasts edit

Chloroplasts are only found in photosynthetic eukaryotes. They convert Sun-derived light energy to ATP and reduced NADPH. Ancient cyanobacteria gave rise to chloroplasts. In other words, cyanobacteria are the ancestor of chloroplasts. Chloroplasts have an outer membrane, an inner membrane, and the thylakoid membrane. Some algal chloroplasts have more membranes outside these. Chloroplasts are typically found within the mesophyll cells, or the inner tissues of a leaf. Chloroplasts are discs that are typically somewhere between 2-7 micrometers in diameter and 1 micrometer thick. Chlorophylls a and b, which are the green pigments located within plant chloroplasts, give plants their typical green color. During photosynthesis, carbon dioxide enters the leaf through microscopic pores known as the stomata, and oxygen leaves as a byproduct through the stomata. The dense fluid found within the chloroplast is known as the stroma, and amongst this are several interconnected membranous sacs shaped in flat discs with their own compartments, known as the thylakoids. In plants, thylakoids are arranged in a stacked conformation known as grana. Most other photosynthetic organisms and some CTemplate:Sub plant chloroplasts have unstacked thylakoids. The space within these compartments are called the thylakoid space. The membranes of the thylakoids hold the cell's chlorophyll.

8. Vacuole edit

Vacuoles serve as the cell's storage centers of food and other necessary materials. The vacuole further functions in the removal of unwanted structural materials, the containing of several waste products and small molecules (which could involve the isolation of potentially harmful products in the cell), the exportation of unwanted materials from the cell, and the maintenance of an acidic internal pH and constant internal pressure. In plants, the central vacuole is typically the largest compartment of all the cell's organelles, occupying in itself the majority of the cell volume. This large compartment is enclosed by a membrane called the tonoplast, which is selectively permeable to certain solutes within the cytosol. The solution inside the vacuole is referred to as cell sap, which is of different composition from the cytosol. Some plant vacuoles contain pigments that color the cells, such as during pollination season by which the plant must attract different organisms to carry out its fertilization. Plants maintain its structure through the maintenance of internal pressure through the manipulation of water into and out of the vacuole. Through water osmosis, water diffuses into the vacuole, which places pressure onto the cell wall. If too much water were to be lost, this pressure against the cell wall would be lacking, and the cell would collapse onto itself. Thus, cells also serve to maintain the cell's size. Another important feature of the vacuole in plants is that an enlarged central vacuole may add a specific amount of pressure against the other compartments in the cell and push them towards the cell membrane, thereby giving a type of conformation that permits the absorbance of more solar energy.

9. Lysosome edit

Lysosomes are membrane enclosed organelles that help eukaryotic cells obtain nourishment from macromolecular nutrients. They contain hydrolytic enzymes. Lysosomal and phagocytosis digestion help the eukaryotic cell because they increase the membrane surface area. In eukaryotes, lysosomes allow intracellular digestion crosses the lysosomal membrane into cytoplasm. Lysosomes are the 'garbage bin' of the cell.

10. Peroxisome edit

Peroxisomes are the centers by which the cell may be rid of potential toxins. The peroxisomes are in itself a receptacle of oxidative enzymes that remove hydrogen atoms from certain organic substances to produce peroxides, which in itself is harmful. This peroxide serves to oxidate the potentially harmful substances, such as alcohol.

Cell Compartment edit

1. Evolution edit

During evolution, cells start to develop into two compartments: the outer and inner aqueous compartment. The advantage of having an inner aqueous compartment allows the better segregation of cellular organelles from the external environment. Thus, each organelle is able to develop and refine its structural and functional distinction inside this aqueous compartment throughout the course of evolution. The "lipid bilayer" (further discussed) existed as a protective wall that allows hydrophilic interaction in the external and internal compartment of the cells while maintain proficient rigidity with its hydrophobic interior structure. The inner aqueous compartment ultimately becomes the cytoplasm which serves the same purpose as it has been during evolution.

2. Liposomes edit

Liposomes are essentially lipid vesicles that are surrounded by a circular phospholipid bilayer. They form identical structure as other phospholipids vesicles: interior hydrophobic tails away from the aqueous solution, and external hydrophilic heads towards the aqueous solution. Liposomes are formed through the process called sonification that results in ions and solutes inside the enclosed compartments. They can be used to study the permeability of certain membranes and transportation of ions or solutes found in different cells.

3. Lipid Bilayer edit

Lipid bilayers form in a spontaneous and self-assembled manner in aqueous environment. Its unique properties allow the formation of enclosed compartments. The sheet-like bimolecular structure called lipid bilayers or energetically favored because of the hydrophobic interactions. As mentioned previously, phospholipids, an amphiphloic moiety as a major class of membrane lipids, exist in an aqueous solution. The hydrophobic tails from two phospholipid bilayers interact with each other to form a hydrophobic center. Meanwhile, the hydrophilic heads line up with each other, form a hydrophilic coating on each side of the bilayer, and isolate the inner compartment from the outer environment.

Membrane Movement edit

A. Lateral Diffusion edit

The lateral diffusion, or the motion of moving laterally, of the biological membranes illustrates the fact that the membranes are not rigid and static. In fact, the membrane is not stable. There is a technique known as Fluorescence Recovery After Photobleaching (FRAP)assists to visualize the lateral diffusion of membrane proteins. An example of the experiment is as follow: 1) Label a specific cell component with fluorescence. 2) Use a intense beam of laser light to bleach, or destroy, the small part of the florescence labeled cell surface. 3) The intensity of bleaching recovers as the lateral diffusion of unbleached membrane proteins move into the region that has been bleached.

B. Transverse Diffusion edit

Transverse Diffusion describes the movement of molecules from one side the membrane to the opposite side. In comparison to the rapid movement of lateral diffusion, the speed of transverse diffusion is rather slow. The reason for the preservation of membrane structural asymmetry is due to the greater energy barriers formed in order to travel across membrane from one side to the other.

The Cycling Process of Cells edit

1. Cells Division edit

Cell division is a process that a cell gets divided and then duplicated. In prokaryotes, cells are divided by binary fission. In eukaryotes, the process becomes more complicated that there are three steps or periods for division. The cell grows during the period of inter-phase, when it absorbs nutrients for mitosis and duplication of DNA. Then the cells comes to mitosis phase. During this mitosis, the cell splits into two different daughter cells. Moreover, the chromosomes in its nucleus will also be divided into two equivalent parts, each into separate nuclei. Finally, the cell finishes division during cytokinesis.

2. Cells Aging edit

Aging is due to continuous damage to various molecules in our cells, including proteins, lipids and nucleic acids. There are internal and external reasons for the damage to happen. The example for external reasons is oxygen, which is regarded as the necessity for human beings. However, when the oxygen absorbs only one or two electrons, making itself reactive, this kind of oxygen molecules will damage lipids, mutate genes, and destroy proteins, all leading to cellular injury. On the other hand, the internal reasons are related to cell retiring. According to the data, cells stop working when they divide about fifty times. This phenomenon might be traced to the period when a cell copies its chromosomes to its daughters. However, the very ends of the chromosomes will not be copied, so daughters’ chromosomes are shorter, compared to their maternal cell. Telomere, at the ends of cells’ chromosomes, supplies the genetic information, which is the same as that on the parts of maternal chromosomes left. Nonetheless, when a cell’s telomeres get shrunk to a minimum size, the cell will stop dividing and lose its function. The picture to the right shows the telomeres in 46 human chromosomes.

3. Cells Death edit

There are two kinds of cell death: apoptosis and necrosis. Apoptosis is programmed cell death that cells that undergoes self-destruction by regulation. At first, a cell shrinks and escapes from its neighbors. Later on, the surface of the cell breaks into fragments and also the nucleus then collapses. In the end, the whole cell disassembles. There will be some organelles cleaning up the remains. During apoptosis, unneeded cells or retired cells are eliminated efficiently without pain. Apoptosis plays a significant role in our body, which is self-destruction. When some cells in our body become infected, apoptosis can help eliminate them to avoid the virus spreading the whole body. However, viruses have several solutions to prevent apoptosis, such as stop cells’ suicide by confusing them with a similar “off” signal as that sent by apoptosis. Therefore, further research in apoptosis can promote the development of clinical medicine. On the other hand, Necrosis is unplanned cell death. It happens when the outer membrane of a cell is unable to control the liquid flowing across it. The cell is then shrink and burst. Finally, contents inside will flow out, blending into the tissues nearby. The reasons causing necrosis might be traumatic injury, infection, chemical poison, etc.

4. Balance in cells edit

Now we understand that some cells are created and some are killed. The truth is that these processes are designed well to keep our body healthy. The unbalance between apoptosis and mitosis will cause cancer.

Endocytosis edit

Endocytosis is the creation of internal membranes from the cell's plasma membrane lipid bilayer. It is a way for plasma membrane lipids and integral proteins to be brought inside the cell. It is thus the opposite of exocytosis. This allows cells to do things like regulate the sensitivity of cells to ligands since receptors can be removed from the cell surface by endocytosis. Plasma membrane buds, called caveolae, are on little creices on the surface of many mammalian cells. These can make up almost a third of a cells surface area. Given their structure, they are often involved in endocytic events. Another way of internalizing in cells is called phagocytosis. In this method, material can be take in when 'invaginations' are formed around particles to be engulfed while using or not using the growth of surrounding membrane extensions.

References edit

  1. Microbiology
  2. Microbiology

1. Berg, Jeremy M. Biochemistry. 6th ed. W.H. Freeman, 2007.
2. Campbell, Neil A. Biology. 7th ed. San Francisco, 2005. 3. "Inside the Cell" of U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES National Institutes of Health National Institute of General Medical Sciences 4. "Mechanisms of Endocytosis". Doherty and McMahon, MRC Lab of Molecular Biology, Cambridge, UK. 5.Inside the Cell, U.S.DEPARTMENT OF HEALTH AND HUMAN SERVICES National Institutes of Health National Institute of General Medical Sciences 6. 7. 8. 9. 10. Slonczewski, Joan L. "Microbiology: An Evolving Science." 2009

The Nucleus edit

Eukaryotes derive their name from the fact that they contain a nucleus. The nucleus is also often the most prominent feature of eukaryotic cells viewed under a microscope. The nucleus is an organelle, an intracellular membrane-enclosed compartment with a specific function. The nucleus also contains chromatin. Chomatin is a complex of DNA and proteins. The nuclear membrane consists two concentric phospholipid membranes. Nuclei contains a region called the nucleolus, where ribosome assembly begins. Besides, the nuclear membrane contains nuclear pore complexes that allow for transport of material into and out of the nucleus. The metabolites and small proteins can diffuse through the NCPs. Large proteins that need to enter the nucleus are actively transported in through the NPCs.

The nucleus is a membrane-enclosed organelle found in eukaryotic cells. It contains the genetic material of the cell, organized as multiple long linear DNA molecules that coil around to form chromosomes. The transcription of RNA also takes place in the nucleus.

Illustration of a human cell nucleus

Function edit

The eukaryotic nucleus contains the genetic information of the cell, and insulates it from the cytosol. It is in the nucleus where the nucleolus is contained; the site of RNA synthesis and DNA replication. During the interphase stage of the cell cycle, the genetic material is found in the form of chromatin. It is during mitosis and meiosis that the chromatin coil together, using histone proteins, to form tightly packed sister chromatids which then replicate, forming daughter cells.

Structure edit

Slide of a nucleus and nucleolus (dark spot)

The nucleus is known as the information storage organelle. it is enclosed by a double membrane- "nuclear envelope". Inside of the nucleus there is DNA. The DNA is bound by proteins- "histones".There are 5 types of histone proteins out of which H2A,H2B,H3 and H4 ,each repeated twice form a core of 8 histone proteins around which wraps the DNA double helix linked by Histone H1 linker DNA proteins.this unit of 8 histones wrapped by DNA is called 'nucleosome'.A repitition of nucleosomes makes a nucleofilament that futher coils to form DNA coils and supercoils that form chromosomes. The dark spot of the nucleus is the nucleolus- site or rRNA synthesis. The nucleus has its own internal skeleton known as the nuclear lamina. It also has its own transport system called nuclear pores.

Nuclear Envelope edit

The Nuclear Envelope is a double layered membrane that surrounds the nucleus. It consist of nuclear pores that regulate the transportation of substances such as RNA into and out of the nucleus. The nuclear envelope consists of an outer nuclear membrane and an inner membrane. Both nuclear membranes are "lipid bilayers". The outer nuclear membrane is extensive and is connected to the rough endoplasmic reticulum or ER. The inner nuclear membrane is continuous with the nuclear lamina consisting of various lamins. The lamina is an attachment site for the chromosomes that also stabilize the structure of the nucleus. The inner nuclear membrane contains different kinds of membrane proteins. The inner and outer nuclear membranes are connected through nuclear pores. There are some bound ribosomes attaching to the outside of the nuclear envelope. These bound ribosomes function on inserting protein into and secreting protein out of the membrane.

Components in a nucleus edit

The cell nucleus is an important organelle because it is where genes and their controlling factors are formed. In order to do so, it needs the helps of the stored components:

  1. Chromosomes: store and organize genes that allow cell division.
  2. Nuclear pores: transport regulatory factos and gene products.
  3. Messenger ribonucleic acid (mRNA): produce messages that code for proteins.
  4. Nucleolus: produce ribosomes that functioning in the expression of gene code into proteins.

Overview edit

The nucleolus, or plural nucleoli, is normally a circular structure composed of proteins and nucleic acids. Nucleoli are not typical organelles for the reason that they have no lipid membrane, making it with of the few non-membrane bound organelles in the cell. The nucleolus is located within the nucleus of eukaryote cells and is in charge of producing ribosomal RNA and the arrangement of ribosomes. The structure of the nucleoli can be seen using electron microscopy and fluorescent protein tagging can be used to view the dynamics of the nucleoli.


Structure edit

The nucleolus has three components:

  1. Fibrillar Centers (FC): FC is the place where ribosomal proteins are made.
  2. Dense Fibrillar Components (DFC): It has new transcribed RNA which binds to ribosomal proteins to form rRNA
  3. Granular Components (GC): Before ribosomes are formed, GC has rRNA that binds to ribosomal proteins.

Function edit

The nucleolus is the nuclear subdomain that assembles ribosomal subunits in eukaryotic cells. The nucleolar organiser regions of chromosomes, which contain the genes for pre-ribosomal ribonucleic acid (rRNA), serve as the foundation for nucleolar structure. The nucleolus disassembles at the beginning of mitosis, its components disperse in various parts of the cell and reassembly occurs during telophase and early G1 phase. Ribosome assembly begins with transcription of pre-rRNA. During transcription ribosomal and nonribosomal proteins attach to the RNA. Subsequently, there is modification and cleavage of pre-rRNA and incorporation of more ribosomal proteins and 5S rRNA into maturing pre-ribosomal complexes. The nucleolus also contains proteins and RNAs that are not related to ribosome assembly and a number of new functions for the nucleolus have been identified. These include assembly of signal recognition particles, sensing cellular stress and transport of human immunodeficiency virus 1 (HIV-1) messenger RNA.

Reference edit

Molecular Picture of a ribosome, Blue = Proteins, Orange = RNA, Red = Active Site

Overview edit

The purpose of the ribosome is to translate messenger RNA (mRNA) to proteins with the aid of tRNA. In eukaryotes, ribosomes can commonly be found in the cytosol of a cell, the endoplasmic reticulum or mRNA, as well as the matrix of the mitochondria. Proteins synthesized in each of these locations serve a different role in the cell. In prokaryotes ribosomes can be found in the cytosol as well. This protein-synthesizing organelle is the only organelle found in both prokaryotes and eukaryotes, asserting that the ribosome was a trait that evolved early on, most likely present in the common ancestor of eukaryotes and prokaryotes. Ribosomes are not membrane bound.

Ribosomes are also play a key role in the catalysis of two important and crucial biological processes. They are responsible for peptidyl transfer, in which they form peptide bonds during protein synthesis as well as peptidyl hydrolysis; in this process, the ribosome releases the completely formed protein from the peptidyl transfer RNA after completion of translation. Translation is the process of converting mRNA (from transcription) to functional proteins; The three steps involved are initiation, elongation, and termination. Proteins are translated one amino acid (three base pairs) at a time. During this process, tRNA assists the ribosome by bringing the complementary bases to the ribosome as translation proceeds.

Because ribosome synthesis is a major metabolic activity that involves hundreds of individual reactions in eukaryotes, errors will occur eventually during these processes. To deal with the error in ribosome synthesis if it occurs, eukaryotes will degrade the erroreous ribosomes. In addition, not only the erroneous ribosomes will be degraded but also the excess ribosomes. Recent researches show that eukaryote cells enhanced many strategies to recognize specific dysfunctional or functionally deficit ribosomes for degradation[1].

Translation: Eukaryotic and Bacterial edit

Along with having more complicated assembly regulations than bacterial ribosomes, eukaryotic ribosomes are triggered differently than bacterial ribosomes in translation initiation. Eukaryotic ribosomes use a scanning mechanism and require at least nine initiation factors. [2] In bacteria, ribosomes can identify the right reading frame and know where to attach to a mRNA stran, by finding the Shine Dalgarno sequence on the mRNA. This ribosome binding site is upstream from the AUG start codon on the mRNA. In the bacterium E. Coli, the Shine Dalgarno site is a purine-rich nucleotide sequence—5’ AGGAGGU 3’, on the mRNA that is located four to eight bases ahead of the start codon. This Shine-Dalgarno sequence is complementary to the sequence 5’ ACCUCCU 3’ on the 16S rRNA, which is found in the 30S subunit of the ribosome. [3]

Structure edit

Ribosomes usually comprise 2 protein subunits and some component of rRNA. The protein componenet of the ribosomes is first synthesized in the cytosol much like any other protein. The newly synthesized protein subunits are then transported from the nucleus into the nucleolus, the center of ribosomal RNA transcription. RNA polymerase are then used to polymerize the rRNA needed for the synthesis of the ribosome. Cells that have a high rate of protein synthesis have many ribosomes.

In the past, scientists studied ribosome to get a better understanding about its components, which is consists of 30S, 50S, 70S, and 100S. All are made of by the same particles but different concentration of Mg ions. 30S and 50S are small and large subunits of 70S while 100S is a collection of 70S. Ribosomal structure varies between eukaryotes and prokaryotes. Eukaryotes have 40S (small) and 60S (large) subunits, together the subunits form the 80S ribosome.[4] Prokaryotes have 30S (small) and 50S (large) subunits. These subunits work together during translation to synthesize proteins.

EColi 70S Ribosome. The red is the 50S large subunit and the blue is the 30S small subunit

In prokaryotes, the small subunit (30S) and the large subunit (50S) together make the 70S ribosome. The 30S subunit is composed of 21 ribosomal proteins and 16S rRNA. Its function is to decode and decipher mRNA to determine the corresponding amino acid to the three bases in codons. The small subunit consists of the body (5’), the platform region (central domain), and the head (3’). Each component can be formed separately of each other. The small subunit also contains a 3’-minor domain consisting of the last two helices (44 and 45) and the end of the 3’ end of the rRNA. The structural components of the small subunit have been shown to be structurally independent from each other suggesting that during protein synthesis they move and conform relative to one another. The long 44 helix stretches across the small subunit from the body to the head functioning as a potential relay for information across the entire subunit. Unlike the small subunit, the large subunit is mostly rigid with mobility restricted its peripheral regions. The 50S subunit consists of 33 proteins and the 23S and 5S rRNA.

The mRNA binding site is located along the neck of the small subunit while the large subunit contains the peptidyl transferase center (PTC) where aminoacyl and peptidyl-tRNA attach. The three binding sites are A, P, and E. Binding site A attracts aminoacyl-tRNA, binding site P attracts peptidyl-tRNA, and binding site E, the exit site, attracts deacylated tRNA. The anticodon stem loop is attached to its complementary codon on the mRNA binding site of the small subunit while the amino acid end of the tRNA attaches to A and P sites both located in the PTC. The GTPase-associated center and the sarcin/ricin loop are located on the large subunit and help to stimulate GTP hydrolysis needed for elongation of the protein.

Ribosome assembly consists of transcription, translation, the folding of rRNA and ribosomal proteins, the binding of ribosomal proteins, and the binding and release of the assembly components to make the ribosome. There are two intermediates in ribosome assembly, in vitro and in vivo. One method of in vitro assembly of the 30S is to only use free rRNA and ribosomal proteins. Another is to combine 16S rRNA with purified and recombinant proteins and precursor 16S rRNA. With the different ways to assemble proteins, the assembly of ribosomes also varies with different temperatures. At low temperature of 0°C -15°C, the reconstitution intermediate (RI) particle, composed of 16S rRNA and 15 ribosomal proteins, is formed and settled to 21S-22S. Then the RI particle is rearranged after getting heated to 40°C and settled to 25S-26S. After completing these two processes, the RI particle together with the remaining proteins can form the 30S subunit.

In contrast, the in vitro assembly of the 50S subunit requires more steps and harsher conditions compared to the assembly mechanism of the 30S. There are three reconstitution intermediates each dependent on different temperatures and ionic conditions. RI50 (1), the first intermediate, yields the 33S particle. As RI50 (1) is heated, RI50 (2) forms and sediments at the 41S-43S. The remaining proteins form the RI50 (3) which create the inactive 48S particle. In order for the 48S particle to become the active 50S subunit, the temperature and concentration of magnesium needs to be increased along with the involvement of the 5S rRNA.

In Vitro and In Vivo Ribosomal Assembly Mechanisms

Like the in vitro assembly mechanism, the in vivo assembly of the 30S subunit has two intermediates (p130S and p230S) and the 50S subunit has three intermediates (p150S, p250S, and p350S). However, the reconstitution intermediates are not the same as in vitro. The intermediates of the 30S subunit yield 21S and 30S particles while the intermediates of the 50S subunit yield 32S, 43S, and 50S particles. The intermediates in the in vivo assembly are precursor rRNA which is different from in vitro which uses matured rRNA. To complete the mechanism of ribosome assembly, these precursor rRNA gets transformed in the polysomes.

Eukaryotic Ribsome edit

A eukaryotic ribosome is composed of a 40S and 60S subunits, they both have a solvent exposed side and a subunit interface. The solvent exposed side contains a higher concentration of protein and RNA elements that are only in eukaryotes compared to the subunit interface.

The eukaryotic specific proteins are responsible for 40S structure being stabilized by tertiary contacts. The majority of the eukaryotic specific proteins and extensions are to interconnect other proteins in the 40S and 60S subunit. The 40S subunit has 14 of these interconnected proteins located in the head. eukaryotic specific proteins and extensions play a bigger role in 60S subunits because their protein mediated connections are more extensive and reach across the subunit. The 60S specific extensions consist of beta sheets and long alpha helices which are crucial in long distance tertierary interaction, meaning they have the ability to interact across the subunit. This trait is unique for eukaryotes because protein-protein contact is very rare in the prokaryote ribosome.

The 40S and 60S subunits are joined by eukaryote specific inter-subunit bridges that connects them thus forming 80s. This is done by eukaryotic specific bridges forming at the 40S and the RPL19 interacts with ES6E and RPL24 interacts with rpS6. The other two eukaryotic specific bridges connect by 60S segment ES31 that connects to rpSA and another 60S segment, ES41 that connects with rp58 from the 40S subunit. [5]

Ribosomal Cofactors edit

Ribosomal cofactors help facilitate with improper folding during in vitro. The improper folding during in vitro is a cause which results in vitro being much slower than in vivo. Ribosomal factors can be used as a “check point” to make sure the assembly is correct. Examples of ribosomal cofactors are DEAD-box proteins, chaperones, and GTPases. DEAD-box proteins are factors which help with proper folding of the RNA. An over expression of DEAD-box proteins can suppress the deletion of another strain. DEAD-box proteins (DBPs) are characterized by a core of approximately 350 amino acids, each containing at least 9 conserved amino acid motifs. Studies have shown that these proteins use the energy from ATP hydrolysis to rearrange inter- or intra-molecular RNA structures or dissociate RNA-proteins interactions. In eukaryotes, DEAD-box proteins are fundamental in the life of a ribosome, but in E. coli, they are often unnecessary. To date, five different genes encoding DEAD-box proteins have been identified in E.coli – SrmB, CsdA, DbpA, RhlE, and RhIB – which are versatile cofactors that have helped further the understanding of cellular processes in many cells because of their roles in protein biosynthesis and ribosome assembly. For instance, CsdA and SrmB are essential for proper cellular growth; cells deficient in these proteins exhibit severe ribosomal effects.

E.Coli Cofactor interaction of various DEAD-box proteins

Brief History edit

Ribosomes played a big role in our evolution. The origin of ribosomes are said to have roots in the RNA world, preceding the existence of proteins. Ribosomes evolved in structure and function, alongside the formation of complex and versatile peptides. A huge breakthrough in the scientific study of ribosomes began when Venkatraman Ramakrishnan and his group determined the crystal structure of the ribosome, and described it in atomic detail. Uncovering the structure of the ribosome allowed scientists a closer look at the mechanisms involved with ribosome function. More specifically for example, according to "The ribosome goes Nobel" article by Rodnina and Wintermeyer, in the "Trends in Biochemical Sciences" journal, knowledge of the structure of the ribosome revealed details of the translation process like "how the ribosome checks for proper codon–anticodon interaction by establishing specific contacts in the decoding site". More recently, ribosomes prove to have huge clinical potential as targets for antibiotic products. With the overuse of antibiotics and antibiotic resistance constantly on the rise, ribosomes could be the basis of new antimicrobials that would retaliate against the infamous antibiotic resistance.

Free Ribosomes edit

Ribosomes found in the cytosol that are not bound to any other organelle are known as free ribosomes. These ribosomes are known to produce non-hydrophobic proteins that ultimately function in the cytosol. An example would be the catalysts for the first steps of sugar breakdown.

Endoplasmic Reticulum Ribosomes edit

Ribosomes which are attached to the endoplasmic reticulum or ERare known as ER Ribosomes. These ribosomes are what give rough endoplasmic reticulum its "rough" appearance. Proteins that are produced from the ribosomes of the rough ER are sent through the lumen of the endoplasmic reticulum, modified within the ER, and then is exported in a vesicle to the cis face of the golgi apparatus, where the protein undergoes further modification.

The Peptidyl-Transfer Reaction Catalyzed by the Ribosome edit

The mechanism of peptidyl transfer catalysis

Enzymes transform their substrates through the active site residues. This transformation can allow for the direct participation in chemical catalysis, such as facilitation of proton transfer reactions and covalent chemistry. These active-site residues can form the covalent bond acting as general acid-base or as nucleophiles. The ribosomes undergo general acid-base or nucleophilic catalysis. The residues can also be structural complement to the transition state (TS), dissolve or reorganize water molecules, and decrease the delta S (entropy) by using the binding energy. In the elongation process, the ribosome PTC catalyzes the aminolysis of the ester bond: the alpha amino group of the A-site aminoacul tRNA acts as nucleophile and attacks the P-site peptidyl tRNA at the carbonyl carbon of the ester bond that connects the tRNA and the peptide. In order to efficiently form peptide bonds amines react with esters. This reaction is fast with the help of the ribosome because it makes the reaction rate increase by approximately 106 - 107 folds. Before the peptide bond is formed, the aminoacyl tRNA connects to the A site at a rate of 10 s-1 which is much slower than the rate of the tertiary complex forming a peptide bond. The slow rate of reaction allows for the minimal use of substrate analog which is short RNA fragments that is similar to the tRNA CCA 3’ end or the alpha amine substituted with a hydroxyl group. The minimal substrates consisted of puromycin (Pmn), C-puromycin (C-Pmn), and CC-puromycin (CC-Pmn). These substrates bind to the ribosome fast to react with the P-site substrate in order to allow for the direct analysis of their chemistry relationship. Recently, methods of analysis using kinetic, biochemical, genetic, and computation allow for more progress in ribosome crystallography. However, there are still many questions regarding the proton transfer mechanism. Peptidyl transfer by the ribosome accompanies a decrease in the entropy activation, which provides more understanding of ribosome catalysis involves defining the molecular mechanism that make entropy of activation lower by taking into consideration the substrate, position of the active site, desolvation, and electrostatic shielding.

1)Peptidyl Transfer 2)Peptide Release 3)Structures of transition state analogs used to study the ribosome

Degradation of erroneous and excess ribosomes in eukaryotes edit

When cells are in stress, disease condition, and normal condition, RNA damage occurs. Exposing to ultraviolet light, oxidation, chlorination, nitration and alkylation result in chemical modifications to nucleobases, generating the potential of triggering ribosome degradation pathways.

Types of error in ribosomes synthesis edit

There are several sources of errors in ribosome synthesis such as the alternation in rRNA sequences (occur in cis) and the failure to bind to or loss of an assembly factor or ribosomal protein (occur in trans). Beside these main sources, specific growth conditions can also cause certain effects in ribosome synthesis. For example, starvation requires excess ribosomes which are turned over efficiently[6].

Potential error-occurring process in Ribosome synthesis edit

mRNA decoding and peptidyl-transfer reaction are two subunites that have specialized function in ribosome translation[7]. Each eukaryote ribosome is composed of 4 rRNAs and ~80 ribosomal proteins. The processes of synthesizing, maturizing, transporting individual ribosomes and assembling into ribosomal subunits requires the contribution of ~200 proteins trans-acting cofactors and a large amount of small nucleolar RNAs (snoRNAs). These processes engage in hundreds of individual error-causing reactions[8][9].In addition, many ribosomal proteins also perform non-ribosomal functions, so the functions in these processes which are not directly connected to ribosome biogenesis are currently being assigned to ribosome synthesis factors (RSFs). RSFs include connections to cell cycle progression, pre-mRNA splicing, DNA damage response, nuclear organizing and telomere maintanance. Due to the large number of reactions that occur in ribosome assembling pathway, the possibility of error occurring is very high such as the potential harms for cell viability and human health. For example, inability to bind correctly to or loss of a synthesis factor can cause the production of erroneous ribosomes such as lacking part of ribosomal proteins or carrying misfolded rRNA which will affect on translation. As a result, cells develop control mechanism to avoid such problems. For example, instead of participating in later steps, synthetic factors that involved in late cytoplasmic ribosome assembly steps can first bind with pre-rRNAs at early stages, bringing pre-ribosomes to productive synthesis pathways[10].

Mutation in cis: non-funtional ribosomal RNA decay edit

There are many pathways that have been known to control the structural integrity of mature RNA molecules[11]. One of the pathways that control the decay of mRNAs is the "no-go" decay (NGD), which the mRNAs that cause the degradation of specific ribosomes[12]. LaRivie`re et al. introduced the way to test whether or not mutations in functional and non-functional ribosomal sites affect rRNA stability which is performing substitutions in the decoding site and peptidyl transferase centre at the position that are essential for ribosomal function in bacteria. This experiment gave result in the identification of non-functional rRNA decay, a pathway that detects and eliminates dysfunctional parts of mature ribosomes[13].

Mutations in trans: The TRAMP-exosome pathway and nucleolar surveillance edit

Aberrant nucleolar and nuclear pre-rRNAs are found to be degraded actively by a nuclear surveillance mechanism that involves the addition of unstructured oligoadenylate tails at the 3’-end of flawed pre-rRNAs. This is done with the TRAMP complexes’ polymerase activity, and after with their degradation by the RNA exosome. TRAMP conplexes consist of a poly(A) polymerase, a zonc-knuckle-containing and putative RNA-binding protein, and the DEVH helicase Mtr4. The pathway goes from having the addition of short poly(A) tails with the actual binding between TRAMP complex and RNA, and these two commit deviated molecules to degradation. This is done by separating them from the normal RNA and by stirring exosomal activity[14].

The decay of bulk ribosome and pre-ribosome by ribophagy and PMN edit

Growth-inhibiting conditions cause ribosome synthesis to shut down. The synthesis of complex molecules from simple molecules such as amino acids and sugars is obstructed and existing pre-ribosomes and mature ribosomes are directed towards bulk degradative pathways. To manage stress, and to adapt to a new environment, pre-ribosomes and mature ribosomes are passed on to be recycled for important cellular components. Ubiquitin, a small regulatory protein essential to 25S NRD (Nonfunctional RRNA Decay), is also responsible for bulk ribosome degradation.[15]

Autophagy is a catabolic mechanism that takes unnecessary or dysfunctional cellular components and performs cellular degradation through the lysosome, or the vacuole in yeast and plants.[16] Starvation conditions cause large portions of the cytosol, along with protein, to assemble and whole organelles, such as ribosomes and mitochondria, are recycled through two major types of autophagy: microautophagy and macroautophagy. Macroautophagy involves the development of a double membrane around the organelle, this together known as an autophagosome. [17] This isolates the organelle and delivers it to the lysosome, which engulfs the organelle through endocytosis for its breakdown and recycling. [18] In contrast, during microautophagy, the lysosome directly engulfs the cytoplasmic material, through the inward folding of the lysosomal membrane. [19] Because lysosomes and vacuoles contain non-specific enzymes for hydrolysis, autophagy can essentially degrade any biomolecule. Macroautophagy and microautophagy are both crucial to a cell's survival during starvation. Both mechanisms associate common trans-acting factors, and both are essentially bulk degradative processes. However, studies show that selective types of both channels exist. [20]

Ribophagy is the process in yeasts in which both small and large ribosomal subunits are are delivered to the vacuole through selective macroautophagy. This process is caused by prolonged nitrogen starvation and specific only to ribosomal subunits. [21] Small and large ribosomal subunit ribophagy depends on different and distinct pathways. The increased turnover kinetics of large subunits are dependent upon the Ubp3 ubiquitin protease and its activator Bre5. Increased turnover rates of small subunit are scantly affected when mutations arise in UBE3 or BRE5, indicating independent pathways of ribophagy. However, for small ribosomal subunit ribophagy, the effector remains unknown.[22] As Upb3 deubiquitylation necessary for 60S ribophagy, cells deficient of Upb3 have an increased ubiquitylation level of many ribosome-associated proteins which have yet to be identified. It is believed that the deubiquitylation of Upb3-Bre5 targets aids autophagosomes with the packing of ribosomes, or allows the maturation of autophagosomes and/or its merging with the vacuole. [23]

Ribosome surveillance in human health edit

Alterations in RNA can cause ribosome degradation, some of which can lead to serious diseases. RNA damage are not only results of stress and disease situations, as they occur during normal cell growth as well. Exposure to ultraviolet light, chlorination, oxidation, alkylation and nitration can all result in the introduction of chemical modifications to nucleobases, as well as RNA-RNA and RNA-protein crosslinks, into RNA and RNPs. [24] Many believe that RNA oxidation could be associated with disease progression, and that many factors, including the extent of association with proteins or iron, may be causes to leaving RNA susceptible to oxidative damage. Some cases of ribosomal RNA damage inflicted by UV radiation and oxidation have lead to human neurodegenerative diseases, such as Alzheimer's, Parkinson's, and atherosclerotic plaques. [25] Reactive oxygen species (ROS) is developed by chronological aging, oxidative stress and other apoptotic stimuli. Yeast cells that are exposed to high levels of ROS fragment Mature 5.8S and 25S rRNA considerably. [26] Yeast cells that are given anti-metabolites and the chemotherapeutic agent 5-fluorouracil (5-FU) accrue polyadenylated pre-rRNAs. In exosome complexes that are hypersensitive to the drug, these accumulations are distressed, proposing that nucleolar surveillance is what is responsible for the degradation of 5-FU. Many nucleolar stresses, such as drug-based interference of pre-RNA processing and rRNA synthesis, have lead to nucleolar breakdown, p53 stabilization and cell cycle advancement defects and/or apoptosis. [27]

Ribosomal Functional Specialization edit

In the core of every ribosome, which is the protein factory, there are ribosomal proteins. Recent research indicates that these core proteins can differ between ribosomes under different growth conditions and that these differences confer the ability to more efficiently translate specific subpopulations of mRNA. Ribosomes exist in every living organism for the same purpose, to synthesize proteins from mRNA. Previously, it was thought that ribosomes were homogenous and lacked any structural diversity. Because function follows structure, these differences should be expected to alter a given ribosome’s affinity for more complementary mRNA strands. In addition to core variants, post-transcriptional modification of the ribosomal RNAs leads to additional structural differentiation, and therefore functional specialization. For ribosomes to be functionally specialized it requires that the producing cell be sensitive to changing environmental conditions such that the ribosomes being synthesized exhibit biochemical differences that affect translation.

References edit

Edward Ki Yun Leung, Nikolai Suslov, Nicole Tuttle, Raghuvir Sengupta, and Joseph Anthony Piccirilli. "The Mechanism of Peptidyl Transfer Catalysis by the Ribosome."

Sebastian Klinge, Felix Voigts-Hoffmann, Marc Leibundgut and Nenad Ban. "Atomic structures of the eukaryotic ribosome." Zahra Shajani, Michael T. Sykes, and James R. Williamson. "Assembly of Bacterial Ribosomes."

Gilbert WV. "Functional Specialization of Ribosomes?" Trends Biochem Sci. 2011 Mar;36(3):127-32. Epub 2011 Jan 16. PMID: 21242088 [PubMed - indexed for MEDLINE]

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  3. Joan L. Slonczewski, John W. Foster. "Microbiology: An Evolving Science."
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  6. Lafontaine, Denis. "Elsevier: Article Locator." | Search through over 10 million science, health, medical journal full text articles and books.. N.p., n.d. Web. 20 Nov. 2012. <>.
  7. Steitz, T.A. (2008) A structural understanding of the dynamic ribosome machine. Nat. Rev. Mol. Cell Biol. 9, 242–253
  8. Henras, A.K. et al. (2008) The post-transcriptional steps of eukaryotic ribosome biogenesis. Cell. Mol. Life Sci. 65, 2334–2359
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  10. Lafontaine, D. et al. (1995) The 18S rRNA dimethylase Dim1p is required for pre-ribosomal RNA processing in yeast. Genes Dev. 9, 2470–2481
  11. Doma, M.K. and Parker, R. (2007) RNA quality control in eukaryotes. Cell 131, 660–668
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Background edit

Ribosome synthesis is a multi-step, error-prone process. The ribosome is comprised of 2 subunits of unequal sizes. These subunits carry out specialized functions within translation such as mRNA decoding for the smaller unit and a peptidyl-transfer reaction for the larger subunit. In eukaryotes, the ribosomes consist of 4 rRNAs and about 80 ribosomal proteins. To synthesis, mature and transport the individual components of ribosomes and assemble them, requires the intervention of approximately 200 protein trans-acting factors as well as numerous amounts of small nucleolar RNAs (snoRNAs). These are involved in the hundreds of individual, error-prone, reactions. There are some functions in processes that don't connect directly to ribosome biogenesis and are then assigned to ribosome synthesis factors such as connections to cell cycle progression, or pre-mRNA splicing and DNA damage response. Because there are so many reactions within a ribosome assembly pathway, the possibility to introduce a mistake with potential deleterious consequences are immense. Such things such as failure to bind or loss of a synthesis factor could lead to a structurally defective ribosome that hold functional consequences in translation. In response to such problems, the cells evolved multiple quality control mechanisms. However, that is not to say that mutations only occur during synthesis as mutations can also occur as a consequence of exposure to genotoxic stress.

Minimizing Defects edit

An example of creating a way to minimize defects would be in a late assembly step, the cell can bind pre-rRNAs at early nucleolar stages thus committing pre-ribosomes to productive syntheses pathways. Another case would be to bind pre-ribosomes to monitor and tether the structural integrity of ribosomal protein-binding sites. This case would be where there are trans-acting factors with partial homology to ribosomal proteins. This is because defects can delay the binding of trans-acting factors.

Mutations edit

Cis edit

Eukaryotes have multiple methods in which they degrade their ribosomes. There are so many reactions in the ribosomal pathways that the possibility of mistakes and mutations are great. Many incidents can occur when mutations occur in cis such as the alteration of rRNA sequencing. Surveillance pathways monitor the structural and functional integrity of RNA. For mutations in the cis conformation, two possible pathways may occur depending on the size and type of the RNA.

There are multiple surveillance pathways that have been described that monitor the integrity of mature RNA molecules, one pathway that monitors mRNA is the "no-go' decay pathway or NGD. This is where mRNAs that induce stalled ribosomes are degraded. LaRivière et al. introduced substitutions in decoding sites and peptidyle transferase center at positions which are necessary in bacteria for ribosome functions. This is done to test if mutations in functionally relevant and conserved ribosomal sites affect the rRNA stability. As a result, the procedure led to the identification of non functional rRNA decay or NRD. In the mRNA NGD, stalled ribosomes triggers initiating endonucleolytic cleavage events on the defective mRNA at the pause site. Then followed by exoribonucleoytic digestion of the 5'- and the 3'- cleaved mRNA products. The RNA exosome digests the defective cleave at the pause site and the 5'->3' exoRNase Xrn1 at the 5'- and 3'- cleaved mRNA. RNA exosome is a conserved multiprotein 3'->5' exoRNase complex active in the synthesis, degradation and surveillance of most classes of cellular RNAs. For NGD, the key components include Dom34 and Hbs1. This however is not the case for 25S NRD, but is true for 18S NRD. As such it indicates two distinct pathways.

In 18S NRD, Dom34 acts together in the same pathway with Hbs1 and they interact in vitro and in vivo. The small molecule inhibitors of translation stabilize the 18S but not the 25S NRD substrates and as a result provides further evidence that 18S NRD activation requires elongating ribosomes and that 18S and 25S NRD are mechanically different. 18S NRD has been linked with cytoplasmic exoRNase Xrn1 and Ski7 as delting both Hbs1 and Ski7 enhances the stabilization of the 18S NRD substrates. Both 18S NRD and mRNA NGD accumulate in the P-bodies that are conserved RNA-protein cytoplasmic granules that contain untranslated mRNA's with a set of repressors. 25S NRD substrates however don't co-localize to P-bodies.

LaRivière's work allowed scientists to discover the "non functional rRNA decay" pathway or NRD. NRD emphasizes on detecting and removing developed ribosomes. NRD is similar to NGD however they both have different kinetics which can be attributed to the higher complexity and compaction of mature ribonucleoprotein particles. NRD has two pathways one in which focuses on the small ribosomal subunits and the other which modifies large ribosomal subunits. There are two main degradation systems for the larger ribosomal subunits: 1. ubiquitin-proteasome system (UPS) and 2. autophagy. UPS targets short lived proteins and involves ubiquitlation and autophagy that degrades long lived proteins.

Trans edit

When errors occur in the trans conformation it can signify a failure to bind to or loss of and assembly factor or ribosomal protein. The TRAMP surveillance revolves around the addition of unstructured oligoadenylate tails at the 3'-end of the destructive pre-rRNAs from a poly(A) polymerase activity. Afterwards this method of tagging leads to the degradation performed by the RNA exosome. The addition of these polyA tails allows one to distinguish between normally functioning RNA and then following with a stimulation by exosomal activity. It is suggested that defected RNAs will go through multiple rounds of TRAMP-mediated polyadenylation and then digested by the exosome to increase the degradation effect. There are certain cases that surveillance takes place in a specialized nucleolar domain called "No-body" that contains much TRAMP and exosomal components.

Pathways of rRNA Decay in Eukaryotes edit

There are five pathways of rRNA decay described to date.(i)Nucleolar and nuclear pre-40S and pre-60S ribosomal units are monitored actively by the "TRAMP-exosome' pathway. If a misfolded pre-ribosomal unit is identified, TRAMP binds to the molecule followed by the polyadenylation of the 3' ends of defective rRNAs in a step that stimulates both the recruitment and decay of the exosome. It is still not understood how TRAMP detects defective ribosomes. Polyadenylation occurs both at normal and cryptic pre-RNA sites. Differences in RNA Polymerase I activity can activate cryptic cleavage sites. Cytoplasmic mature subunits carrying cis mutations are monitored by the NRD pathway.(ii) During 18S NRD, small subunits with errors along the mRNA are identified by Dom34 and Hbs1. These errors are cleaved endonucleolytically by an unknown activity (thought to be similar to mRNA NGD). The releases products are digested by Xrn1 and the RNA exosome assisted by its cofactor Ski7. (iii) During 25S NRD, defective 60S subunits are targeted for proteasomal degradation by Rtt101-Mms1-mediated ubiquitylation of unidentified associated ribosomal components. During conditions of starvation in the cell, excess ribosomes are turned over to ribophagy and PMN. (iv) Ribophagy is a type of macroautophagy that involves the engulfment of cytosolic fractions to the vacuole. Inside the vacuole, the components are recycled. (v) In PMN, a specific type of microautophagy, a portion of the nuclear envelope is pinched off by the vacuole. This creates a specialized organelle called the NVJ that matures into a vesicle. Finally, the mature vesicle is degraded by resident hydrolases.

Ribosome surveillance in human health edit

RNA damage occurs under normal cell growth as well as during stress and in disease situations. Chemical modifications to nucleobases are introduced into RNA and RNPs from exposure to ultraviolet light, oxidation, chlorination, nitration, and alkylation. These alterations all constitute potential physiological triggers to ribosome degradation pathways. Neurodegenerative diseases such as Alzheimer's and Parkinson's have been correlated to damage in rRNA sequences because of exposure to UV light or oxidation. RNA oxidation has been speculated to be involved in disease progression and that RNA susceptibility to oxidative damage is influenced by various factors including the degree of association with protein protection. Mature 5.8S and 25S rRNA are fragmented heavily in yeast cells exposed to high levels of a reactive oxygen species generated by oxidative stress (including exposure to hydrogen peroxide and menadione), chronological aging, and other apoptotic cues. Yeast cells treated with the anti-metabolite and chemotherapeutic agent 5-fluorouracil (5-FU) accumulate polyadenylated pre-rRNAs. This accumulation is exacerbated in exosome mutants, that are hypersensitive to the drug. This suggests that the degradation of 5-FU containing RNAs happens through nucleolar surveillance. Nucleolar dysfunction in cells has been correlated with cancer. Various nucleolar stresses such as drug mediated interference of rRNA synthesis, pre-rRNA processing, or inhibition of ribosome synthesis factor function, lead to nucleolar disruption. Ultimately, the cell cycle becomes defective. An interesting regarding this phenomena is the observation of an increased abundance of polyadenylated rRNA fragments in the gut of western honey bees infected with colony collapse disorder (CCD). The insect guts serves as a primary interference with the environment taking in all the pesticides from outside. CCD has been linked to picorna-like viral infections known to hijack cellular ribosomes. This in addition to the use of pesticides could trigger a ribosome degradation response.

Concluding Remarks edit

Ribosome synthesis is a major cell activity that can enforce quick energy drain with little regulation. Control is exerted at the level of synthesis, assembly of the pieces, and surveillance of the final product. Ribosome synthesis has evolved to be fully integrated with complex nutrient sensing mechanisms. In the prescence of defective ribosomes, damaged ribosomes, or abundance of mature ribosomes, they are targeted for rapid breakdown and recycling. Many surveillance pathways exists that either select excess or defective ribosomes. The pathways are even intricate enough to survey large or small subunits of the ribosome. The most important concept is that we understand how all these pathways interconnect in the ribosome's synthesis.

References edit

Lafontaine Denis L.J. "A 'garbage can' for ribosomes: how eukaryotes degrade their ribosomes." <>

Intro edit

Ribosomes are present in both eukaryotic and prokaryotic cells. However between the two exists differences in the functions and characteristics of the ribosomes. In eukaryotic cells ribosomes are assembled in the nucleus and transferred to the cytoplasm where they finish maturation. Maturation includes trans acting shutting factors, transport factors, incorporating the rest of the proteins in ribosomes, and the final step in rRNA step process. Recent research, for example on the large ribosomal subunit has confirmed that 60S subunit is transported from the nucleus using an inactive state. After the subunits reaches the cytoplasm it leads to events that cause it to be transitionally competent.

Biogenesis edit

In cells the ribosome is responsible for the last step in decoding information from genes in proteins. Ribosomes are made of subunits, which are complex and made of RNA and proteins. The two subunits each have a distinct job they are responsible for. Small 40S subunit is used to decode and the large 60S is responsible for polypeptide syntheses. Using structural analyses of prokaryotic ribosomes provides detailed insights for the mechanisms of the ribosome functions, however what we know about the vivo assembly of ribosomes is still not as well known and is still rudimentary. Biogenesis begins with the transcription of pre-RNA that undergoes co-transcriptional folding, modification and assembly with r-proteins forming the two subunits. In bacteria assembly of the subunits need a few trans-acting factors. However in eukaryotic cells it is a complicated process which requires all three RNA polymerase and over 200 trans acting factors. Thus helping the maturation and intercellular transport if the subunits. Ribosomes in eukaryotic cells are assembled in the nucleolus at the rRNA transcription. The released pre-ribosomes although seeming preassembled required maturation steps in the nucleoplasm and the cytoplasm. In the 1970s Planta and Warner's work led to the identification of the first pre-ribosome, 90S particle. 90S is processed to give the smaller 66S and 43S particles. These being the precursors to the mature 60S and 40S subunits. The particles contain pre-rRNA, r-proteins and various amount of trans acting factors. In the early 1990s applying genetic approaches in buddying yeast allowed for the identification of various trans acting factors which led to a better and more clear understanding of the high ordered steps in the processes involved for rRNA. Even though these advances were accomplished, the composition and make up of pre-ribosomal particles still remains unknown and a mystery until the recent decade. With tandem affinity purification, TAP, in combination with mass spectrometry has allowed us to isolate and analyse the composition of maturing pre 60S and pre 40S in buddying yeast.The analyses helped in the ordering of the pre-ribosomal particles in the 60S and 40S pathways, allowing us to picture the highly complex assembly process.

In yeast, RNA polymerase I in the nucleolus helps transcribe 35S. The transcribed rRNA is methylated, pseudo-uridylated, and loaded with r-protein and trans acting factors which allows it to form 90S. 90S particle has r-proteins and trans acting factors which are important for the 40S biogenesis pathway. The cleavage of the rRNA at the A2 site thus releases the pre 40S particle which the maturation and biogenesis are independent of the 60S. Once pre 40S is released the remaining pre-rRNA assembles with large subunit r-proteins and the biogenesis factors form the pre 60S particles. Differently, pre 40S particles undergo few compositional changes as they move from the nucleoplasm. Compared to the pre 60S, pre 40S are exported to the cytoplasm. In contrary pre 60S particles are associated with about 100 trans acting factors along the biogenesis pathway and also change in composition as they move through the nucleoplasm to the nuclear pore complex.

In biogenesis there are different stages that take part in the nucleus and the cytoplasm. In the stages the trans acting factors are released from pre-ribosomal particles and are recycled for the new rounds of biogenesis. The events are caused by enzymes that consume energy that are associated with maturing pre-ribosomal particles. The site of these events of the enzymes and the precise function of them in ribosome maturation still remains unknown to us. Research in the field has shown that two large AAA-ATPases Rix7 and Rea1 implicates in maturation of the pre 60S subunit. Rix7 seems to strip Nsa1 fromt he subunit in the nucleolar transition, and Rea1 is believed to drive pre 60S particles to export competence by the removal of Rsa4. The AAA-ATPases is this believed to contribute directly to the sequential reduction of the complexity of the pre-ribosomal particles, before they are removed from the nucleus.

Nuclear export of pre-ribosomal subunits edit

Once pre-ribosomal subunits are produced in the nucleus, they are transported into the cytoplasm through the NPC (Nucleaer Pore Complex). The NPC is the largest protein complex in the cell an dis responsible for the protected exchange of components between the nucleus and cytoplasm. It also serves to prevent the transport of material not destined to cross the nuclear envelope [1]. It is found that for the nuclear export of r-subunits, unique nucleoporins are needed to make the export of both subunits to happen. Nucleoporins are simply the proteins of the NPC. Also, researcher have discovered that Ran GTP-GDP cycle is required. Pre-40S and pre-60S particles are exported independently of each other, however, they both need to have the general nuclear export factor Xpo1 or Crm1 that directly recognizes nuclear export sequences.[2] Nuclear export sequences are amino acid sequenecs that label a protein for export into the cytoplasm from the nucleus. Nmd3 is the only known Crm1 adapter for the pre-60S particle. However, there are at least three NES-containing trans-acting factors that function as Crm1 adapters in pre-40S export. Those trans-acting factors include Ltv1 (in humans), DIM2 and RIO2.[2] An interesting fact is that there is an redundancy in 40S export adapters as the nature of Ltv1 is almost non-essential. In order to achieve efficient export of pre-ribosomal subunits requires multiple receptors. For example, in budding yeast, pre-60S particles use additional factors that assist the nuclear pore complex for its export business.

Maturation of pre-ribosomal subunits at the cytoplasmic level edit

During the early stage of biogenesis, many of the trans-acting factors that are related with pre-ribosomal particles are released from the nucleus and can be recycled back. This process happens before nuclear export. There is a few factors remain related to to the pre-ribosomal subunits as they go into the cytoplasm. We will look at the maturation of the pre-60S subunit and pre-50S subunit independently.

Maturation of the pre-60S subunit edit

Pre-60S suniunits enter the cytoplams with a entourage of non-ribosomal factors that must be released by unique factors in the cytoplasm. It should be emphasized that several ribosomal proteins are needed to add functionality to the subunits. The steps in the maturation of the pre-60S subunit are shown as below.

  1. Pre-60S particles exit the nucleus and enter the cytoplasm
  2. In the cytoplasm, a third essential AAA-ATPase is introduced to pre-60S particles

Maturation of pre-ribosomal subunits edit

The trans acting factors that are associated with pre-ribosomal particles in early biogenesis are released and recycled to the nuclease before nuclear exportation, but some factors are still associated with the particles as they enter the cytoplasm. By releasing and recycling the factors as well as the assembly of the remainder of the r-proteins and the finals processing of RNA constitute the "cytoplasmic maturation steps" in the ribosome biogenesis pathway. The steps needed are not only crucial for complete maturation of subunits but also because if it fails to recycle a factor the nucleus it leads to its depletion from the nucleolar, thus inducing lays in pre-rRNA processing, defects in assembly, and impaired nuclear export.

'Pathway' of cytoplasmic maturation edit

ATPases and GTPases do not dependent on each other and therefore the events driven by ATPases and GTPases can occur without a specific order. However, some evidence shows that coupling of these events could happen. Drg1 a type of AAA-ATPase, blocks Rlp24, Nog1, and Arx1 from recycling and also blocks Tif6 from recycling to an extent. With this information, it is possible to start ordering the events. First, Drg1 releases Rlp24, and the loading of Rlp24 into subunits brings Rei1. Arx1 is then released when Rei1 works with Jjj1 and Ssa1/Ssa2. Arx1 being in the subunit eventually hinders Tif6 from being released. Starting with Drg1 releasing Rlp24, a series of events takes place and eventually it gets to the release of Tif6.

Conclusion edit

Since it is extremely crucial that translation of the genetic code done correctly, it is reasonable to speculate that quality-control mechanisms are evolved by eukaryotic cells to monitor ribosome biogenesis. Few research proved that cytoplasmic maturation steps in 40S and 60S biogenesis pathways are done by activation of subunits. This is accomplished when inhibitory factors are removed and functionality is added. Transfer of only the functional ribosomal subunits is guaranteed with the control of these cytoplasmic maturation steps.

References edit

  2. a b Panse, Vikram Govind; Johnson, Arlen W. (May 2010). "Maturation of eukaryotic ribosomes: acquisition of functionality". Trends in Biochemical Sciences. 35 (5): 260–266. doi:10.1016/j.tibs.2010.01.001.

Overview edit

Ribosomally synthesized natural products (RNPs) are a class of peptides that are of interest to scientists because of their great diversity courtesy of post-translational modifications (PTMs) like hetero- or macrocy-clization, dehydration, acylation, glycosylation, halogenation, prenylation, and epimerization. These RNPs have great potential because PTMs with respect to structure and biological activity compared to the traditional 20 amino acids. They are also favorable because RNPs have relatively short biosynthetic pathways and like to interact with numerous other compounds.

Lantipeptides edit

A group among RNPs that is of particular interest is the lantipeptides. They are polycyclic peptides that contain the thioether cross-linked amino acids meso-lathionine (Lan) and (2S,3S,6R)-3-methyllanthionine (MeLan). Lantipeptides are all synthesized on the ribosome as a precursor LanA peptide, which contains both a leader peptide, the N-terminal portion of the precursor peptide important for production, but unmodified and cleaved from the final product and a core peptide, the C-terminal portion of the precursor peptide that becomes the final product after modification and leader peptide removal. There are four classes of lantipeptides: class I, class II, class III, and class IV. Class I lantipeptides perform serine/threonine dehydration and thioether cyclization by the dehydratase LanB and the cyclase LanC, respectively. Class II lantipeptides have LanM, a single bifunctional lantipeptide synthetase containing N-terminal dehydratase and C-terminal LanC-like cyclase domains. Class III lantipeptides are modified by a trifunctional synthetase with an N-terminal lyase domain, a central kinase domain, and a putative C-terminal cyclase domain. Finally, class IV lantipeptides have LanL, which contains N-terminal lyase and kinase domains as in class III, but its C-terminal cyclase domain is analogous to LanC.

Lantipeptides were originally thought to be only produced by a specific group of gram-positive Firmicutes, but genomic analysis has shown that other organisms also produce lantipeptides as well like actinomycetes, bacteroidetes, and chlamydiae. A recent study identified lantipeptide biosynthetic genes in 478 of 1,466 bacterial genomes. This shows how abundant lantipeptides are in biological organisms and can be found and harvested from a pool of organisms for researchers to use to do experiment involving these products.

Lantipeptides are interesting to scientists because they have antimicrobial properties, but researchers are finding numerous other uses for them as well. For example, there is interest in using lantipeptides as chemotherapeutics. This idea came from the use of the lantipeptide nisin as a food preservative for more than 50 years because of its use has not presented significant microbial resistance.

Lantipeptides also have a lot of potential in the pharmaceutical industry. They have been proven effective against gram-positive bacteria, including drug-resistant strains like Staphylococcus, Streptococcus, Enterococcus, and Clostridium, and certain gram-negative pathogens like Neisseria and Helicobacter. A few drugs made from lantipeptides are duramycin for the treatment of cystic fibrosis and a derivative of actagardine for the treatment of Clostridium difficle Mutacin 1140 is currently in the developmental stages for the treatment of gram-positive bacterial infections. Other than pharmaceuticals, lantipeptides have applications in agriculture, veterinary medicine, and molecular imaging, proving their diversity in biochemical activity.

Researchers have studied the mechanisms by which lantipeptides conduct biological activity and it has been observed that most antibacterial lantipeptides inhibit cell wall biosynthesis and/or disrupt membrane integrity through pore formation. Lantibiotics, lantipeptides with antimicrobial activity, target and bind to the essential cell wall precursor lipid II. This inhibits the reaction required to synthesize peptiodglycan, an essential part of the cell membrane.

The unique thioether cross-links of lantipeptides are added on post-translationally by biosynthetic enzymes via dehydration of serine or threonine to the α,β-unsaturated residues 2,3-didehydroalanine (Dha) or (Z)-2,3-didehydrobutyrine (Dhb), respectively, followed by Michael-type addition of a cysteinyl thiol to give a thioether bridge. The cross-links are essential for the activity of the compound and for maintaining stability against proteolysis and heat denaturation. This makes lantipeptides quite durable in biological conditions that would otherwise denature other peptides.

Class I edit

In order to synthesize class I lantipeptides, two separate enzymes carry out reactions forming thioether cross-links: dehydratase LanB and the cyclase LanC. LanB genes encode proteins of about 1,000 residues, while the lanC genes encode proteins of approximately 400 residues. It is believed that these reactions involve a zinc ion bound by a cysteine-cysteine-histidine triad at an active site.

In Vivo Engineering edit

Currently techniques are being developed to improve the pharmacological properties of lantipeptides, including potency, stability, and solubility. One approach is in vivo lantipeptide production in E. coli. This simplifies genetic manipulation and cuts costs because E. coli is relatively easy to grow and is a well-studied organism. A 2011 study demonstrated the versatility of E. coli production of modified LanAs. This study also represents the only report to date of class I lantipeptide production in E. coli. Two other E. coli expression systems reported in 2011 have utilized dedicated lantipeptide proteases to produce mature lantipeptides. One system produced a fourfold improvement in yield compared to the producing strain. Using E. coli appears to be a viable in vivo method for producing lantipeptides and further research will likely yield more effective methods.

Chemical Synthesis edit

In addition to using biological techniques to acquire lantipeptides, chemical methods have shown some promise as well. Before 2008 the only successful total chemical synthesis of a lantipeptide was the impressive solution-phase synthesis of nisin. However, recently the first first solid-supported total synthesis of a lantipeptide containing overlapping cross-links, the α-peptide of lacticin 3147, has been reported. This expands the possibilities for synthesizing lantipeptides and should be studied further in conjunction with bioengineering techniques.

References edit



Introduction edit

Algal cells contain chloroplasts, membrane enclosed organelles of photosynthesis that evolved from a cyanobacterium. In Earth's biosphere, algae plus bacterial phototrophs feed all marine and aquatic ecosystems, producing the majority of oxygen and biomass available for Earth's consumers. Besides, the "green plants" include primary endosymbiont algae descended from a common ancestor containing a chloroplast. The chloroplasts of primary endosymbionts are enclosed by two membranes. They are the inner membrane and the outer membrane. The inner membrane is from the ancestral phototroph's cell membrane and the outer membrane is from the host cell membrane as it enclosed its prey. The chloroplast of the green algae and red algae have diverged from their common ancestor that use the pigments absorbing different ranges of the light spectrum.


Chloroplasts are organelles responsible for photosynthesis or the conversion of CO2 to glucose in plant cells, some protists, and algae. There are three types of membranes in chloroplasts:

  1. The smooth outer membrane that is permeable to molecules
  2. The smooth inner membrane contains membrane proteins that selectively allow access to small molecules and proteins
  3. The thylakoid membrane system

The fluid inside the chloroplast and surrounding the thylakoid system is known as the stroma.

Thylakoid edit

Thylakoid membranes envelop a system of interconnected vesicles. Stacks of these thylakoid membranes are known as grana. The main function of the thylakoid membranes is to house the following protein assemblies:

  1. Photosystem I
  2. Photosystem II
  3. Cytochrome b and f
  4. ATP synthase

All of these carry out photosynthesis as a whole.

The thylakoid membranes also contain chlorophyll, which gives plants its green color. The function of the chlorophyll is to capture the light necessary for photosynthesis.

In the thylakoid membrane and compartment, the light reactions take place in the thylakoid membrane and the thylakoid compartment and are concerned with the initial conversion of light energy into chemical energy that stored in ATP and NADPH.

Besides, the ATP and NADPH feed into the Calvin cycle in the Chloroplast stroma where the ATP provides energy for the molecular rearrangments and also the electrons carried by the NADPH are transferred to the organic molecules involved in the Calvin cycle.

Inhibition of photosynthesis edit


There are many herbicides on the market that advertise for their ability to kill weeds. In order to kill the weeds, a disruption of photosystem I or photosystem II is required. The basic idea is that the electrons are activated once they received sunlight. The activated electrons then moves from the chlorophyll into a series of cytochromes creating what is called an electron transport chain. These electrons that are transferred along the electron transport chain are later used for carbon fixing, hence resulting in photosynthesis. An interruption in photosystem II is a result of the inhibitors stopping the flow of electrons, whereas an interruption in photosystem I is initiated by an inhibitor that creates change in energy through electron diversion at the terminal photosystem. Both types of interruptions can stop the process of photosynthesis.

Some inhibitors of photosystem II are diuron and atrazine. Diuron blocks the active site of plastoquinone in photosystem II, and its blockage disrupts the steady flow of electrons to the plastoquinone. As a result, sunlight cannot be converted to energy, and ultimately leading to the death of weeds. Atrazine works in similar ways as diuron. An example of photosystem I inhibitor is paraquat. Paraquat converts the electrons that it receives from photosystem I to radicals. The radical then interacts with oxygen to form reactive oxygen substances. These substances, in turn, interact with the membrane lipids, which can cause damage to the membrane because the double bonds of the membrane lipids are altered.

Stroma edit

The stroma is the fluid contents of the chloroplast, it contains the enzymes of the chloroplast such as RUBISCO, which is responsible for the dark reactions. The stroma is also home to the DNA of the chloroplasts that helps it to carry out its functions, some ribosomes of its own, and RNA. Due to the chloroplasts’ individual DNA, it has been hypothesized that the chloroplasts were once free living bacteria that became involved in symbiotic relationship with eukaryotes and eventually became permanently incorporated into the cell’s structure. [12]

The Dark reaction, mainly the Calvin Cycle that take place in the substance surrounding the thylakoids. Carbon from the carbon dioxide is brought into the cycle as a source of carbon in the Calvin cycle.

Purpose edit

Chloroplasts produces starches and sugars. In order to do so, the plants extract energy from the sun. The energy extraction from the sun is called photosynthesis. With the energy extracted, the chlorophyll in the plants can combine carbon dioxide and water together. Both the animals a plants uses chloroplast for energy and food. Animals also uses it for oxygen to breath.

  • Molecular Form: 6 CO2 + 6 H2O --> sugar(C6H2O6) + 02

Reference edit

  2. Berg, Jeremy "Biochemistry", Chapter 27 the Integration of Metabolism. pp 584. Seventh edition. Freeman and Company, 2010.

Slonczewski, Joan L. Microbiology "An Evolving Science." Second Edition.

Amino Acids edit

Proteins have a lot of uses in the body. from cell structure to carrying out reactions. Proteins are in turn made of smaller building blocks called amino acids. There are a total of 20 amino acids.

The 20 Amino Acids

Proteins can be made from a lots of different combinations and number of amino acids. This creates an almost infinite amount of different proteins. Although the sequence of amino acids is a chain, the structure of proteins is far from similar. These amino acids interact with each other to form #D structures. Most common are the alpha helix and the beta strand.

A picture of an alpha helix
Amino acids interacting to make a beta strand

Although it is possible to determine the 3D structure of a protein, it is not quite possible to predict protein structure from the amino acid chain. This is quite important because diseases such as Alzheimer's and Mad Cow Disease are thought to come from a misfolding of proteins. [1]

References edit

  1. The Structures of Life, NIH Publication No. 07-2778

Function edit

1. Nuclear semi permeable membrane 2. Nuclear pore 3. Rough endoplasmic reticulum (REM) 4. Smooth endoplasmic reticulum 5. Ribosome attached to REM 6. Macromolecules 7. Transport vesicles 8. Golgi apparatus 9. Cis face of Golgi apparatus 10. Trans face of Golgi apparatus 11. Cisternae of Golgi apparatus 12. Secretory vesicle 13. Cell membrane 14. Fused secretory vesicle releasing contents 15. Cell cytoplasm 16. Extracellular environment

Endoplasmic Reticulum is an extensive membranous network in the eukaryotic cells that is continuous with the outer nuclear membrane and composed of ribosome-studded (rough) and ribosome-free (smooth) regions.

The ER is responsible for the manufacture of the membranes and performs many other biosynethic functions.

The endoplasmic reticulum (also known as the ER) is made up of a wide system of membranes that make up over fifty percent of the total membrane in numerous eukaryotic cells, and consists of two sections that have different functions: the smooth endoplasmic reticulum and the rough endoplasmic reticulum. The endoplasmic reticulum consist of a system of membranous tubules and pouches called cisternae, which is held together by the cytoskeleton. The membrane segregates the inside section of the endoplasmic reticulum from the outside section (cytosol) . The internal section is called the lumen cavity, but is also known as the cisternal space. The name “endoplasmic” is defined as “within the cytoplasm,” and the Latin definition for the word “reticulum” is “little net.” The membrane of the endoplasmic reticulum is continuous throughout the nuclear envelope. Because of this, the area between the two membranes of the envelope is also continuous to the cisternal space.

The Endoplasmic Reticulum contains a quality control mechanism called chaperones. These special proteins attach themselves to newly synthesized proteins and assist them in folding into their native conformations. Charperones also keep mis-folded proteins in the ER to be broken down. These incorrectly folded proteins are degraded through the process of ER Associated Degradation (ERAD). These Chaperones can be affected by genetic diseases such as Cystic fibrosis. Cystic fibrosis is a genetic disease that causes the buildup of chaperones and their incorrectly folded proteins in the ER. This disease leads to mucus clogging the lungs and digestive track, which can lead to early death.

The ER Under Stress edit

Under stress conditions, the normal function of the ER is impaired, causing the alteration of protein homeostasis and the corresponding accumulation of misfolded proteins in the ER lumen. This condition is known as 'ER stress'. The aglomeration of misfolded and/or unfolded proteins, detonates a dynamic response process called 'unfolded protein response' (UPR), which is the combination of several intracellular signaling pathways that have as a main purpose to reduce the load of misfolded proteins in order to re-establish protein homoestasis, adjusting to the current need in a dynamic fashion. In addition, the UPR carries out the transcriptional induction of genes expressing chaperones, ER-associated degradation (ERAD) components and autophagy regulators to the cytosol and the nucleus. If the upregulation of proteins involved in folding, quality control and trafficking is not enough to balance ER homoestasis, the UPR eliminates the damaged cell by apoptosis.

UPR Sensors edit

The UPR (mentioned above) is activated by three stress sensors, that detect the accumulation of misfolded or unfolded proteins in the ER. These sensors are:

  • Inositol-requiring enzyme 1α (IRE1α): It's a type I transmembrane protein with an N-terminal luminal region, a cytosolic kinase and RNase domains. Its activation has been explained by two different proposed models. The first one explains that immunoglobulin-binding protein (BiP), an ER-resident chaperone, inhibits oligomerization of IRE1α by binding to it. However, the aggregation of misfolded proteins causes BiP to dissociate from IRE1α, allowing it to interact with unfolded proteins and thus, to oligomerize. The second model affirms that unfolded proteins interact directly with IRE1α's luminal domain, inducing its oligomerization. These mechanisms are still under debate, although recent research data leads to the idea that both of them operate together in the regulation of IRE1α. The oligomerization process undergone by IRE1α leads to to trans-autophosphorylation of its kinase region which in turn engages its RNase activity. Activated IRE1α launches alternative splicing of the mRNA that encodes the transcription factor X-box binding protein 1 (XBP1), leading to the translation of a more stable spliced form of XBP1 (XBP1s), which in turn upregulates certain UPR-related genes associated with folding, ERAD and quality control.
  • Protein kinase RNA-activated (PKR)-like ER kinase (PERK): It's a type I transmembrane protein that contains both a cytosolic kinase domain and an N-terminal luminal stress sensing domain. Its activation is carried out by similar mechanisms to those of IRE1α. Activated PERK contributes to reducing overload synthesis of proteins in the ER.
  • Activating transcription factor 6 (ATF6): It's is a type II transmembrane protein, which under basal conditions associates with BiP. The accumulation of unfolded proteins initiates the dissociation of ATF6 from BiP, leading to the translocation of monomeric ATF6 to the Golgi and finally to the nucleus, where it upregulates the transcription of ER chaperones and ERAD-related genes.

Four Steps: Adaptation to Apoptosis edit

The activation of the UPR Sensors is followed by a Four Step process in which the cell first attempts to repair the stressed ER. If this fails the cell will set itself on a path to apoptosis.

Step One edit

The First Step of the process attempts to decrease the amount of unfolded proteins on the ER. This is done by activating the previously introduced Protein kinase RNA-activated (PKR)-like ER kinase, or PERK. PERK directly decreases the amount of all protein synthesis in an effort to halt the production of proteins that are difficult to fold. Additionally Inositol-requiring enzyme 1α (IRE1α) plays a role in this stage by initiating macroautophagy, a process to destroy misfolded proteins to relieve stress on the ER.

Step Two edit

The Second Step consists of a focus on the increase in quality of proteins being produced and restoring homeostasis to the ER. This is accomplished by two methods in no particular order. First, transcription factors XBP1s, ATF6f, ATF4, AXP1s, and ATF6f collectively work to folding and degradation capacity by upregulating chaperone activity,protein modification enzymes and the size of both the ER and Golgi. The second part of this step is ATF4 upregulating genes that change the redox state of the ER to provide a better environment for the folding of proteins. Both of these steps work to reduce the amount of misfolding proteins and increasing the precision with which new proteins are constructed to avoid producing more misfolded and unfolded proteins.

Step Three edit

The Third Step begins to transition the cell to a state where apoptosis is more likely. This step has two contributing factors, both influenced by a protein called C/EBP homologous protein or CHOP. CHOP, acting downstream of AFT4 changes the redox state of the entire cell so that the cell is out of its natural state and therefore has trouble functioning. Additionally, again after attempts to slow protein synthesis, CHOP increases the protein synthesis of the cell further stressing it. Both of these factors will sensitize the cell to apoptosis.

Step Four edit

The Fourth Step is the apoptosis of the cell. When ER stress results in apoptosis, the BCL2 protein family acts to bring the cell to apoptosis; all pro-apoptotic members of this family contain BH1, BH2, and BH3. Specifically, BH3 acts to trigger the activation of BAX and BAK. These then permeablilize the membrane of the cells mitrochonira and release cytochrome c and apoptosome. This moves the cell into a stage of apoptosis.

ER Stress and Illness edit

When discussing ER stress and human disease, it is important to recognize that nearly all data that suggests a linkage between ER stress is either correlative or based on in vitro evidence. Correlative evidence is flawed as it is unwise to assume that correlation always implies causation, and in vitro data is flawed as it is an isolated study rather than a comprehensive view of the organism in question.

Tumors and Cancer edit

Tumors often cause hypoxia in the tissues they affect. When this occurs, the ER begins a stress response to adapt to the new conditions. Under normal conditions the efforts of the ER stress response would be beneficial to the body, however, the result of the adaptation is that the tumorous cells are allowed to replicate, driving tumor growth rather than the death that would have occurred had ER stress not took place. Scientists believe that the IRE1α-XBP1 pathway involved in this process is a promising target for cancer treatment.

Diabetes edit

When glucose levels become excessive, an ER stress response is initiated. This response is thought to suppress insulin gene expression via IRE1α degradation of insulin mRNA. This downregulation of insulin is characteristic in type two diabetes.

The article "Endoplasmic Reticulum Stress and Type 2 Diabetes" by Sung Hoon Back and Randal J. Kaufman, they discuss how, due to certain lifestyles now, type 2 diabetes has vastly increased over the years. Type 2 diabetes is "characterized by increased levels of blood glucose owing to insulin resistance in adipose tissue, muscle, and liver and/or to impaired insulin secretion from pancreatic [beta]-cells." They discuss how beta-cells increase in response to obesity and insulin resistance, but it doesn't last forever and normally begins to fail. It is possible that beta-cell loss could be due to endoplasmic reticulum (ER) stress. Pancreatic beta-cells secrete insulin, which is a "blood glucose-lowering hormone." Proinsulin is synthesized in the ER and then stored in beta-cells until glucose levels increase and then insulin is released. The ER is in charge of many things and requires a certain environment, so when the environment changes, there are several stress responses that occur to try to keep the ER functioning properly. The main stress response is called the unfolded protein response (UPR). UPR includes "expansion of ER size, enhanced folding capacity, reduced protein synthesis through transcriptional and translational controls, and increased clearance of unfolded or misfolded proteins." IF this does not work and the ER does not return to homeostasis, then cell death is normally activated. It is believed that the ER sends signals for apoptosis is the stress is severe and unresolved by UPR. IRE1α can also mediate cell death pathways. ER stress can increase activation of IRE1α which leads to increased splicing of genes leading to cellular dysfunction and beta-cell death.

Neurodegenerative Diseases edit

Correlative evidence shows that ER stress markers are present in deceased persons that suffered from Alzheimer's, Parkinson's, ALS, Huntington's, and Creutzfedt-Jabob disease. Scientists recognize however, that the role of ER response will be neurodegenerative diseases will be very difficult to determine.

Final Remarks edit

The detailed study of persistent ER stress and the UPR is essential for the complete understanding of certain diseases, such as cancer, diabetes, neurodegenerative diseases (including AAlzheimer’s, Parkinson’s and amyotrophic lateral sclerosis) and inflammation-related diseases. What such diseases have in common is the accumulation of abnormal intracellular and/or extracellular protein aggregations and inclusions. Therefore, by the meticulous study of the enzymatic pathways followed by the UPR stress sensors, new pharmachological interventions could be developed for the mentioned illnesses.

Rough Endoplasmic Reticulum (RER) edit

The description of endoplasmic reticulum (ER) being "rough" comes from the organelle being studded with ribosomes. These ribosomes on the rough ER, are used to synthesize proteins which are destined to either be inserted into the membrane of the cell or transported out of the cell. Differences between ribosomes lying on the ER and those lying in the cytosol stem mainly from the destination the protein that is synthesized by the ribosome. With protein synthesis via ER ribosomes, proteins are synthesized as they normally would through translation of mRNA. After the protein is synthesized, called "posttranslation", it then enters the lumen of the endoplasmic reticulum, the inner compartment of the organelle. Although the secondary structure (i.e. beta sheets and alpha helixes) of the newly-synthesized peptide forms almost instantaneously as it is translated,its tertiary structure does not begin to form until it is in the lumen, where it will begin to fold into its specific 3-D conformation with the aid of chaperone proteins [1]. It is inside the lumen of the rough endoplasmic reticulum that N- and O- linked glycosidic bonds are formed between amino acids and sugars, thus this process is called "glycosylation" and the newly formed protein is called "glycoprotein".

Vesicle Transport edit

The endoplasmic reticulum transports the proteins through the use of vesicles. Vesicles are membrane enclosed packages that contain protein. On the surface of the vesicles, there are coating proteins called COPI and COPII. The protein COPII allows the vesicles to be sent the Golgi and the COPII protein allow the vesicles to come back to the Rough ER. From inside the Golgi, proteins are again packaged into vesicles and transported to the cell membrane. Once at the membrane, the vesicle itself merges with and become part of the membrane as it is composed of the same lipid bi-layer. Its protein content either will remain on the membrane if it is to function as a membrane protein or be secreted outside of the cell [1].

Smooth Endoplasmic Reticulum (SER) edit

The smooth endoplasmic reticulum, unlike its "rough" counterpart, does not package protein. Instead it carries out various metabolic reactions within the confines of its membrane. Functions range from carbohydrate metabolism, lipid synthesis (for example oils, new membrane phospholipids, and steroids), and even drug detoxification. Drug detoxification is usually accomplished by adding hydroxyl groups to drug molecules. This makes them more soluble in water and therefore easier to be flushed from the body. Another important function of the smooth endoplasmic reticulum is the storage of calcium ions. This is imperative for muscle contraction. When a muscle cell is stimulated by a nerve impulse, the calcium ions rush into the cytosol and trigger muscle contraction.

Structurally the smooth ER is not continuous with the cell's nuclear envelope. The smooth ER is involved in phospholipid synthesis.


Interesting Facts about the Endoplasmic Reticulum edit

1. Proteins are folded and modified in this organelle of the cell.

2. The internal volume of lumen in the endoplasmic reticulum (ER) is almost equal to the outside of the cell. This supports the idea that the ER evolved from the cellular membrane.

3. The ER contains an environment that is distinctly different from that of the cytosol in terms of solute and protein composition. This is because this environment is conducive to the ER’s role as a “staging post”; proteins stop at the this organelle to be packaged and modified before exiting the cell. The ER is also used as a checkpoint before the proteins are secreted, checking the protein quality and accuracy in terms of folding and modification before the protein is allowed to exit the cell.

4. How proteins are folded and modified within the ER: When a protein first enters the ER, it is recognized by a signal sequence typically found at the N terminus of the protein. This sequence is recognized by a receptor located on the surface of the ER membrane called signal recognition particular (SPR). The molecule is then directed towards a pore, called the Sec61 translocon, in the membrane of the ER where it is allowed to pass through and into the ER. One interesting fact about protein folding is that when some proteins are passed through the ER, before entering the ER lumen, they are mere polypeptide chains. The folding of the protein actually sometimes happens entirely within the ER lumen! It is therefore important to note that when the polypeptide sequence enters the lumen of the ER and are subject to the modifications of the sidechains of its amino acids, this can alter the way the protein would fold. Thus, these modifications influence folding, and folding sometimes also influences modifications too. There are some exceptions to this, such as transmembrane proteins that have large cytosolic domains. These proteins fold within the lumen and within the cytosol of the cell. Once the protein is fully translocated into the ER lumen, it was finish the folding and modification that it needs. If the protein is part of a multisubunit complex, then the ER will assemble the protein into its native oligomeric structure. Modifications catalyzed by enzymes in the lumen of the ER greatly aid the maturation of the protein and bring it closer to its final desired outcome. Once this is complete, the finished protein is then put into a transport vesicle, which buds off from the ER and continues on its journey to either the golgi apparatus or for secretion. Some proteins, which do not end up being modified correctly can be consequently targeted for destruction and may leave the ER to be degraded by enzymes in the cytosol.

5. The UPR (mentioned above) is activated by three stress sensors, that detect the accumulation of misfolded or unfolded proteins in the ER. These sensors are:

  • Inositol-requiring enzyme 1α (IRE1α): It's a type I transmembrane protein with an N-terminal luminal region, a cytosolic kinase and RNase domains. Its activation has been explained by two different proposed models. The first one explains that immunoglobulin-binding protein (BiP), an ER-resident chaperone, inhibits oligomerization of IRE1α by binding to it. However, the aggregation of misfolded proteins causes BiP to dissociate from IRE1α, allowing it to interact with unfolded proteins and thus, to oligomerize. The second model affirms that unfolded proteins interact directly with IRE1α's luminal domain, inducing its oligomerization. These mechanisms are still under debate, although recent research data leads to the idea that both of them operate together in the regulation of IRE1α. The oligomerization process undergone by IRE1α leads to to trans-autophosphorylation of its kinase region which in turn engages its RNase activity. Activated IRE1α launches alternative splicing of the mRNA that encodes the transcription factor X-box binding protein 1 (XBP1), leading to the translation of a more stable spliced form of XBP1 (XBP1s), which in turn upregulates certain UPR-related genes associated with folding, ERAD and quality control.
  • Protein kinase RNA-activated (PKR)-like ER kinase (PERK): It's a type I transmembrane protein that contains both a cytosolic kinase domain and an N-terminal luminal stress sensing domain. Its activation is carried out by similar mechanisms to those of IRE1α. Activated PERK contributes to reducing overload synthesis of proteins in the ER.
  • Activating transcription factor 6 (ATF6): It's is a type II transmembrane protein, which under basal conditions associates with BiP. The accumulation of unfolded proteins initiates the dissociation of ATF6 from BiP, leading to the translocation of monomeric ATF6 to the Golgi and finally to the nucleus, where it upregulates the transcription of ER chaperones and ERAD-related genes.

References edit

Braakman, Ineke, and Neil J. Bulleid. "Protein Folding and Modification in the Mammalian Endoplasmic Reticulum." Annual Review of Biochemistry 80.1 (2010): 110301095147058. Print.

Woehlbier, U., Hetz, C. Modulating stress responses by the UPRosome: A matter of life and death. Trends in Biochemical Sciences, June 2011, Vol. 36, No. 6.

Back, Sung H., Kaufman, Randal J. "Endoplasmic Reticulum Stress and Type 2 Diabetes." Annu. Rev. Biochem. 2012. 81:767–93

Endoplasmic Reticulum Stress and Type 2 Diabetes edit

Introduction edit

The Endoplasmic Reticulum (ER) is an important organelle in the cell that performs a wide variety of tasks including and not limited to protein folding, modification, and transfer of membrane proteins to and from Golgi apparatus. Also, the maintenance of ER homeostasis in insulin-secreting beta-cells is extremely important, because when the ER’s homeostasis is disrupted, the ER generates an unfolded protein response (UPR), to maintain homeostasis of this organelle. However, if homeostasis is not restored, the ER promotes death signal pathways. Observations have shown that type 2 diabetes is an important causal factor in the disruption of ER homeostasis and subsequent death of insulin secreting beta cells.

The frequency of Type 2 diabetes in society has dramatically increased due to unhealthy living and eating lifestyles with the consumption of foods rich in fat. Type 2 diabetes consists of a mixture of metabolic conditions characterized by increased levels of blood glucose. The disease develops in areas of the body that are resistant to insulin. Also it is important to note that the disease develops only when beta-cell dysfunction appears. Blood glucose and fatty acids that cannot be properly stored or excreted can overload the cell and disrupt ER and mitochondrial functions. High glucose and saturated fatty acids cause beta cell failure and subsequent oxidative stress through its metabolism.

ER Stress by Lipotoxicity edit

Elevated levels of plasma free fatty acids (FFA) are associated with high fat diets and obesity in humans. Free fatty acids are considered to be vital mediators of apoptosis and dysfunction of pro insulin beta cells in type 2 diabetes. It is known that both unsaturated and saturated FFas inhibit proinsulin synthesis. The precise mechanism of Beta cell dysfunction and apoptosis is not yet fully understood. There is great evidence of long saturated fatty acid chains being responsible for the induction of ER stress and subsequent beta cell failure.

ER Stress by Glucotoxicity edit

Insulin deficiency associated with insulin resistance is known to cause blood glucose levels to remain high (hyperglycemia). Over stimulated beta cells show a gradual decrease of glucose-induced insulin secretion and insulin gene expression and eventually impaired beta-cell function and survival. This phenomenon is known as gucotoxicity. As blood glucose levels continue to remain high, the glucose that originally served as fuel is now used to generate detrimental metabolites for beta cells.

References edit

1. Endoplasmic reticulum stress and type 2 diabetes. Back SH, Kaufman RJ. Golgi apparatus, also known as the Golgi complex, golgi body, or simply golgi, is an organelle found in most eukaryotic cells. The golgi can be thought of as the "packaging, modifying and shipping warehouse" of the cell.

Overview edit

Micrograph of Golgi Apparatus.

The Golgi apparatus, also called the Golgi complex, is commonly found in eukaryotic cells. The Golgi complex can be identified by its unique structure which some say looks like a maze, but in fact the structure is made of stacks of flattened membranous sacs, or cisternae. Unlike the cisternae of the endoplasmic reticulum or ER, these membranes are not connected. The Golgi apparatus is responsible for the processing and packaging of protein and lipids, as well as processing proteins for secretion. The Golgi apparatus is often thought of as a post office; through the process of protein glycosylation, sugars are added to the protein which dictate to where the protein should travel. N-linked glycosylation is begun in the endoplasmic reticulum but is continued in the golgi complex. O-linked glycosylation is solely done in the golgi complex. After proteins have been modified in the golgi apparatus, they are usually sent to either the lysosomes, secretory granules, or plasma membrane depending on the signals encoded within the protein sequence and structure. For this reason, Golgi complex is recognized as "the major sorting center" of the cell.

Golgi Apparatus as an Independent Organelle edit

Researchers in Yale show that the Golgi Apparatus is not just an outgrowth of another organelle, the endoplasmic reticulum, but an independent organelle on its own. The Golgi Apparatus is an organelle within a cell that has its own autonomy and so must grow and divide to keep pace with the growth and division that the cell inhibits. It has been found that even without the stacks of membrane compartments where secretory proteins pass, the Golgi Apparatus still functions. Proteins referred to as matrix proteins that form a scaffold have been found to be responsible for its growth, division and partitioning.

The Golgi Apparatus has been observed to grow, divide and be inherited like other organelles as has been observed in the regrowth of the mother Golgi Apparatus in daughter cells with the use of matrix proteins.

History edit

The Golgi was discovered by Italian physician Camillo Golgi in 1898 during an investigation of the nervous system. [1] Although the discoverer denoted the structure as the "internal reticular apparatus," scientists changed it to the "golgi complex" in 1910. Few doubted the discovery, claiming that the organelle was a mere illusion created by the optical instruments used that discovered them. Yet, soon after the advent of modern microscopes circa 1900's, scientists alike confirmed the presence of the Golgi.

Structure edit

The Golgi complex is made several flattened membranes sacs, but can be ultimately divided into two sections: the Cis Golgi and the Trans Golgi Network (TGN). The Cis Golgi functions as the receiving end for newly synthesized proteins from the lumen of the Endoplasmic Reticulum (ER). Vesicles containing proteins from the ER merge with the Cis golgi allowing the proteins to enter the Golgi complex. As the Cis golgi receives proteins from the ER, the proteins then begin their modification moving along membrane to membrane towards the TGN. At the other end of the golgi complex, the newly modified protein arrives at the TGN where it is then send off to different parts of the cell via a transport vesicle.

Origin of Golgi Apparatus edit

There are two prevailing theories as to the formation of the Golgi apparatus. The vesicular shuttle model postulates that Golgi cannot be made from scratch and that the vesicles of the endoplasmic reticulum are sent to the pre-existing Golgi. On the other hand, the cisternae maturation model suggests that vesicles from the ER fuse together to form the Golgi and as proteins are processed and mature they create the next Golgi compartment. New data suggest that perhaps neither model is completely correct. This will likely lead to yet another model. [2]

Cisternae Maturation Model edit

Cisternae Maturation Model shows that proteins from the ER join to make parts of the Golgi, so the Golgi can be made.

On the other side of the debate is Jennifer Lippincott-Schwartz of the National Institute of Child Health and Human Development (part of the National Institutes of Health) in Bethesda, Maryland. She says that the Golgi makes itself from scratch. According to her theory, packages of processing enzymes and newly made proteins that originate in the ER fuse together to form the Golgi. As the proteins are processed and mature, they create the next Golgi compartment. This is called the cisternae maturation model.

In this model, the cisternae of the Golgi apparatus move by being built at the cis face and destroyed at the trans face. The vesicles from the endoplasmic reticulum fuse together to form a cisterna at the cis face and this cisternae would appear to move through the Golgi stack when a new cisterna is formed at the cis face. This model is supported by the fact that structures larger than the transport vesicles were observed microscopically to progress through the Golgi apparatus. In summary, packages of processing enzymes and new proteins originating in the ER fuse together to form the Golgi and as the proteins are processed and mature, the next Golgi compartment is created. Vesicular transport is one of models of Golgi apparatus that says that Golgi cannot make from scratch and that vesicles in the ER are sent to pre-existing Golgi. [3]

Vesicular Shuttle Model edit

On one side of the debate is Graham Warren of Yale University School of Medicine in New Haven, Connecticut, who argues that the Golgi is an architectural structure that cannot be made from scratch. He believes that newly made proteins are packaged in the rough ER and are sent for further processing to a pre-existing structure (the Golgi) that is made up of different compartments. This is called the vesicular shuttle model.

This model views the Golgi apparatus as a very stable organelle that is divided into compartments in the cis to trans direction. Membrane bound carriers transport material between the endoplasmic reticulum and the different compartments of the Golgi. Experimental evidence shows the abundance of small vesicles in close proximity to the Golgi apparatus. Actin filaments direct the vesicles by connecting packaging proteins to the membrane to ensure that they fuse with the correct compartment. To summarize, newly made proteins are packaged in the rough ER and are sent for processing to a pre-existing structure known as the Golgi which is made up of different compartments.[4]

Vesicle Transport edit

As soon as modified proteins reach the Trans Golgi Network (TGN), the proteins are separated into several different transport vesicles for different areas of the cell.

Vesicle Type Description
Exocytotic vesicles These vesicles contain protein that are to be sent outside the cell membrane. These vesicles fuse with the plasma membrane, releasing their contents outside the cell. This process is called constitutive secretion.
Secretory vesicles These vesicles are also to be sent outside the cell membrane. However what differs these from exocytotic vesicles is that secretory vesicles need a signal before they are released. When the signal is given, they will fuse with the plasma membrane to release the contents. This process is called regulated secretion.
Lysosomal vesicles These vesicles contain protein that are sent to the lysosome to be digested.

Transport Mechanism edit

The transport mechanism that proteins use to progress through the Golgi apparatus is still not clear yet, but there are a number of hypotheses that currently exist. Up until recently, the vesicular transport mechanism had been the favored mechanism for transport but there is now more evidence that supports the Cisternal maturation. The two models may work in conjunction with one another rather than being mutually exclusive. This is sometimes referred to as the combined model.

Glycosylation edit

This is the process of adding sugar to a molecule. Glycosylation happens in the golgi apparatus and in the endoplasmic reticulum. Glycosylation can only happen when an amino acid group with a hydroxyl (-OH) attaches to a sugar molecule. Through a condensation reaction (water leaving) a sugar is added to the amino acid.

References edit

  1. Berg, Jeremy M., John L. Tymoczko, and Lubert Stryer. Biochemistry. 6th ed. New York: W.H. Freeman and Company, 2007. Print.
  3. Inside the cell, U.S. Department of Health and Human Services
  4. Viadiu, Hector
  5. Reece, Jane (2011). Biology. Pearson. ISBN 978-0-321-55823-7. {{cite book}}: Text "coauthors+ Lisa A. Urry, Michael L. Cain, Steven A. Wasserman, Peter V. Minorsky, Robert B. Jackson" ignored (help)
  6. Machalek M Alisa. "Inside the Cell" The National Institute of General Medical Sciences (2010): 36-37.
  2. U.S. Department of Health and Human Services. Inside the Cell. September 2005.<>.
  3. U.S. Department of Health and Human Services. Inside the Cell. September 2005.<>.
  4. U.S. Department of Health and Human Services. Inside the Cell. September 2005.<>.

Function edit

Mitochondria seen through an electron microscope

The purpose of the mitochondria in the eukaryote is to provide cellular respiration to the cell. The endosymbiotic theory asserts that the mitochondria came to be part of the eukaryote over time through a symbiotic relationship. The mitochondria consists of two membranes, the inner membrane and the outer membrane. It is speculated that the outer membrane came about when its ancestor was engulfed by the host celled via endocytosis, giving it a membrane in addition to the one the mitochondria ancestor already had. This endosymbiont theory would also explain why the mitochondria had its own DNA and why this DNA is circular. For some amino acids the genetic code of the mitochondria differ slightly from that of the nucleus (and the rest of the cell). The mitochondria's energy from respiration is stored in an ion gradient across the organelle's double membrane, known as the "Mitchell Hypothesis".

Mitochondrial Biogenesis: It’s a metabolic adaptation where mitochondrial mass increases which allow for conduction of glycolysis, oxidative phosphorylation, and ultimately result in higher mitochondrial metabolic capacity. With greater capacity to synthesize and transport fuels to mitochondria, the metabolic response would be faster, which can be beneficial for athletes during exercise. However, this improvement requires exercise and training.

Mitochondria produce ATP by oxidative respiration. A cell may contain tens or hundreds of mitochondria, depending on its energy needs. Mitochondria have two membranes: an outer membrane and an inner membrane. The inner membrane has many infoldings called cristae that increase its surface area. It is thought that as a result of the endocytosis of the bacterium, the inner mitochondrial membrane is derived from the larger eukaryote. Also, the inner membrane has structural characteristics of a prokaryotic cell membrane, while the outer membrane is similar to the host eukaryotic membrane. Mitochondria contains two distinct compartments: the matrix inside the inner membrane and the intermembrane space between the two membranes. The tricarboxylic acid cycle takes place inside the matrix, the metabolic equivalent of the prokaryotic cytoplasm. Furthermore, the fact that the mitochondria contain their own DNA and ribosomes lends further support to endosymbiosis, since these cell features would be a necessary part of any free-living organism. Both the DNA and ribosomes of mitochondria show similarities with the DNA and ribosomes of bacteria.

Inner Mitochondria & Matrix edit

Inside the deepest compartments of the mitochondria is the mitochondrian matrix. It is in the matrix that cellular respiration occurs, where pyruvate (a product of glycolysis in the cytosol) is converted to Carbon Dioxide and water. The matrix is the site of the citric acid cycle, whereby the electron transport chain is used to setup a proton gradient between the inner and outer membrane of the mitochondria, known as the inter membrane space. The protons in the inter membrane space accumulate to a point that the concentration gradient causes the protons to flow back into the matrix.

It is the inner membrane that is studded with the proteins necessary for the electron transport chain, such as the cytochrome electron shuttles. Upon reentering the matrix, the H+ go through ATP synthase, which in turns powers the synthase to phosphorylate adenosine diphosphate (ADP) to adenosine triphosphate (ATP). The ATP can be used later on to be coupled with thermodynamically unfavorable reactions to allow those chemical reactions to proceed. The inner membrane is folded and convoluted which allows for a greater surface area to utulize for the electron transport chain. These convolutions are what make up the cristae.

Interestingly enough the matrix of the mitochondria are one of the few locations outside of the nucleus where genetic information can be found in the cell. Mitochondrial DNA is similar in appearance to that of bacterial DNA due to its circular shape. The matrix is also known to house tRNA and ribosomes, which further solidifies the theory that the mitochondria entered the ancestral eukaryotic cell as single celled organism.

Outer Membrane edit

The outer mitochondria membrane consist of a phospholipid bilayer], laced throughout with integral proteins. The lipid bilayer contains porins which allow the passage of molecules which are 10,000 Daltons or less. This permeability in the outer membrane allows for water, ions, and some proteins to flow freely into the inter membrane space.

Mitochondria as ATP Consumer edit

It is well documented mitochondria produce the ATP necessary for life, and that a proton gradient and membrane potential are required in order for ATP synthesis to occur in the mitochondria. However, mitochondria is very dynamic in that it can reverse its process. Complex V is the enzyme responsible for final ATP synthesis at the end of oxidative phosphorylation. When the trans-membrane potential is insufficient for ATP production, complex V, or F1F0-ATP synthase, can reverse its process, instead hydrolyzing ATP in order to pump protons out of the mitochondrial matrix in order to restore the proper gradient.

In a normal mitochondria performing respiration, ATP is removed through the adenine nucleotide translocase, or ANT. This helps maintain the trans-membrane potential, favoring phosphorylation of ADP. However, ATP hydrolysis is favored when the ANT is reverse, shuttling in ATP from glycolysis.

The consumption of ATP by mitochondria can clearly be potentially lethal to cells in conditions of extreme proton gradient degradation. It can also serve as a potential mechanism used by pathogens in the case of diseases related to mitochondrial respiration inhibition, (such as lack of oxygen in stroke or heart attack, or in less extreme cases where mitochondria are affected such as Alzheimer’s or Parkinson’s).

This reversal process of complex V is directly affected by IF1, the inhibitory factor of F1F0-ATPase. In response to acidification of the matrix of the mitochondria, the IF1 protein inhibits the activity of complex V as ATPase, usually in conjunction with the halting of respiration in conditions such as hypoxia or ischaemia. There is still much to learn about the mechanisms of IF1, however it is the major factor in protecting cells from ATP depletion in hypoxic conditions.

The crystal structure of IF1 is known. The protein acts as a homodimer, inhibiting two F1-ATPase units synchronously. There are many residues in the protein complex that form many associations with subunits of the F1-ATPase. It is believed that full association of IF1 with the F1F0-ATPase occurs only during ATP hydrolysis. It is suggested that IF1 may loosely bind to the F1-ATPase even during normal respiration, and that it may also aid in the efficiency of oxidative phosphorylation by serving as a ‘coupling factor.’

Mitochondria:ROS and Authophagy edit

The site of Reactive Oxygen Species production with in the cell is found in the mitochondria. Reactive oxygen species (ROS) are small, highly reactive molecules that are short-lived. An incomplete one-electron reduction of oxygen is how ROS is formed.ROS includes oxygen anions, free radicals, and peroxides. Autophagy is one of the signaling pathways of the redox regulation of proteins by levels of ROS. Stress conditions activate autophagy, but pathological conditions deregulate autophagy. A build up of ROS can lead to oxidative stress that causes cellular constituents to be oxidized and damaged. Non-enzymatic and enzymatic antioxidizing agents have been created by the cell to prevent oxidative stress. Antioxidants are natural downregulators of ROS inducing autophagy. Autophagy is inhibited by TIGAR.

Role in Signaling Pathway

In addition to being known for their toxic effects on the mitochondria, ROS are also found to help in cellular signaling pathways. For example, when the brain and body are low on oxygen (hypoxia), mitochondrial generation of ROS to turn on signaling pathways that regulate transcription, maintenance of calcium stores, and overall energy management. At the level of the organism, ROS helps to control and manage fluid absorption and exchange of oxygen in the pulmonary circuit. Studies have shown ROS to help with the level of cellular signaling but more research still needs to be done to determine if whether ROS processes are innate to the mitochondria or if other cellular factors are required.

Source: Mitochondrial reactive oxygen species regulate cellular signaling and dictate biological outcomes Robert B. Hamanaka and Navdeep S. Chandel1,2,* 1 Department of Medicine, Division of Pulmonary and Critical Care Medicine, Northwestern University Medical School, Chicago, IL 60611, USA 2 Department

Reactive oxygen species edit

Although reactive oxygen species (ROS) is commonly viewed as toxic byproducts of mitochondrial synthesis of ATP, ROS are crucial as intermediates of cellular signaling pathways; ROS has essential role in oxidative homeostasis and propagation of signaling pathway. There are certain pathways that affect or depend on the production of ROS. The level/accumulation of ROS also suggests biological outcomes.

How ROS are produced?

  • ROS are produced during electron transport chain (ETC) of mitochondrial synthesis of ATP
  • Through complex I, II, and III of ETC, molecular oxygen is reduced to superoxide anion, which is primary ROS produced by mitochondria.
  • ROS in the matric are produced through all complex I, II, and III
  • ROS in the intermembrane space are only produced through complex III
  • The rate of production of ROS depends on the concentration of electron carriers that are able to reduce molecular oxygen and type of electron carriers, which have different rate of electron release and supply among different electron carriers.

How level of ROS responses to hypoxia and mediates signaling pathways?

  • Under hypoxia, cells are exposed to low oxygen. Consequently, adaptive transcriptional program, reduction of cellular oxygen usage, and decrease in the consumption of ATP are promoted by the activation of signaling pathways due the increase in production of ROS.
  • In response to hypoxia, the production of ROS is increased through Q-cycle, which is a specific hypoxic generation of ROS through mitochondrial complex III. The increase in ROS then activates the signaling pathways.
  • Increase in production of ROS inhibits the activity of PHD. The inhibition of activity of PHD later enables the stabilization of HIF that eventually leads to the transcriptional regulation, such as erythropoiesis, glycolysis, angiogenesis, cell cycle, and survival
  • Increase in the production of ROS triggers the activity of AMPK that increase the production of ATP and minimize the cellular usage of ATP. The activation of AMPK also inhibits the activity of mTOR so that ATP is conserved by inhibiting protein translations that consumes lots of ATP. Eventually, the increase in the production of ROS also reduces the consumption of oxygen because the accumulation of ATP is maximized by activity of AMPK

How level of ROS responses to PI3-kinase pathway?

  • The activation of PI3 leads to the activation of Akt that would increase the accumulation of ROS by two ways.
  1. Through metabolic pathway, mTOR is activated by Akt activity, and oxygen consumption and production ATP are increased. As a result, the production of ROS is increased
  2. Akt activity inhibits ROS scavenging by inhibiting FOXOs, which are antioxidants that inhibit the activity of ROS

Important role of ROS

  • Stem cell population
  • Low level of ROS leads to quiescence, which is a state of inactivity, and maintenance of stem cell population. While the level of ROS increasing, population of stem cell is differentiating and proliferating
  • Oxidative homeostasis
  • ROS regulates activity of phosphatases that oppose the activity of kinases and possess a reactive cysteine, which enables oxidation of cysteine and the reduction of ROS
  • ROS maintains oxidative homoeostasis through regulating phosphatases
  • TNFα treatment
  • Outcomes of TNFα treatment depends on activity of two TNFR complexes
  • NF-ĸB → cell survival
  • JNK → cell death
  • The activity of TNFR complexes depend on cellular level of ROS
  • Increasing level of ROS → increasing activity of JNK and decreasing activity of NF-ĸB → cell death
  • Decreasing level of ROS → decreasing activity of JNK and increasing activity of NF-ĸB → cell survival

How ROS affects the cellular transformation?

  • Cellular transformation is caused by the activation of oncogenes and loss of tumor suppressor genes. As a result, there are signaling pathways controlling proliferation, survival and metabolism of cancer cells.
  • In normal cells, high level of ROS triggers the activation of tumor suppressor to enable the apoptosis and senescence
  • High level of ROS → high mutation rate and genomic instability → tumor growth
  • ROS as signaling intermediates: if Ras or Myc is expressed due to certain accumulation of ROS, the cellular transformation will occur.
  • Vicious cycle
  1. lose of tumor suppressor
  2. sustainability of higher level of ROS
  3. proliferation, angiogenesis, and survival pathway of tumor
  4. more accumulation of ROS
  5. leads to #1
  • High level of ROS not only promote tumor growth but also increases possibility of metastasis, the spread of cancer cells from one part to another non-adjacent part

Misconception about ROS

  • Misconception #1: ROS are solely damaging agents
  • It is wrong because only when the accumulation of ROS is really high, the irreversible damages (cellular transformation, tumor growth, etc.) will occur. In fact, certain amount of ROS is required for the cell to proliferate, differentiate, and promote the fitness of the organisms.
  • Misconception #2: Because ROS contributes to human aging and pathologies, mortality can be increased through scavenging ROS by antioxidants.
  • It is not true because study has shown there is a correlation between lifespan extension and high oxidative stress. This correlation suggests the association between lifespan extension with increase in mitochondrial metabolism and production of ROS

Quality control edit

Reactive oxygen species (ROS), the byproducts of synthesis of ATP in mitochondria, is harmful; ROS damage molecules in mitochondria; as a result, mitochondria are no longer functional. The accumulation of molecular damage would finally lead to cellular degeneration and death. The solution of the accumulation of ROS in mitochondria is the quality control (QC) mechanisms that keep mitochondria functional. Although different QC pathways minimize the harm of ROS in different ways, each QC pathway has its own capacity of regulating the ROS. Therefore, the mitochondrial QC work into a hierarchical surveillance network.

QC at molecular level

  • ROS scavenging
  • The first defense in a network of QC pathways
  • Counteract molecular damage when a critical threshold of molecular damage is reached
  • Key components: small molecules and enzymes
  • Decelerate the speed of molecular damaging
  • Although ROS scavenging pathways are effective in decelerating the space, they are unable to completely prevent molecular damage
  • Repair and Refolding
  • The second defense in a network of QC pathways
  • Repair specific modifications and restore the function of impaired function after damage has occurred
  • The homeostasis of mitochondria protein is controlled through protein degradation, de novo protein synthesis, and refolding of misfolded proteins back to their original 3-D structure
  • The repair of damaged mitochondrial DNA (mtDNA), which encodes essential subsets of proteins, the two RNA subunits of the mitochondrial ribosome and tRNAs needed for mitochondrial protein biosynthesis, is also important
  • Base excision repair
  • Direct reversal
  • Mismatch repair
  • Proofreading activity of polymerase during mtDNA replication
  • However, majority of proteins are not able to repair or refold efficiently.
  • Removal and replacement
  • The defense in a network of QC pathways after the repair and refolding of proteins
  • A molecular pathway of removing aberrant protein through protein degradation machinery when a critical threshold of unfolded or damaged protein is reached
  • Due to the limited capacity of repair and refolding of proteins, there is further decline of cellular function, removal and replacement of damaged proteins through proteolysis
  1. Degradation of damaged proteins; cleavage of presequences and the maturation of proteins
  2. replacement with newly synthesized functional proteins

Even though mitochondrial QC in molecular level can somewhat regulate the damage due to ROS, it is not sufficient enough to keep mitochondria functional and proliferating over time. Since several mitochondrial protein complexes are also encoded by nuclear genome, after the removal of damaged proteins, the coordinated expression of mitochondrial and nuclear genes and correct assembly of the proteins into macromolecular complexes is also essential for keeping mitochondrial functional. This last crucial step is dependent on the import of proteins from cytoplasm and a correct inner-mitochondrial sorting.

QC at the organellar level

  • Fission and fusion of mitochondria
  • The first QC pathway in organgllar level when the accumulation of damage molecules can not be solved by molecular QC pathways
  • Fission – content separation
  • Separation of larger organelle into multiple smaller organelles
  • Controlled by three proteins: Dnm1, Fis1 and Mdv1
  • Fusion – content mixing
  • Combination of multiple smaller organelles into larger organelle
  • Controlled by three proteins: Fzo1, Ugo1 and Mgm1
  • Through fission and fusion, mitochondria are able to maintain their functions due to degradation of damaged mitochondria; each mitochondria wouldn’t have too much damaged components since content are either mixing or separating from each other.
  • High stress → fission
  • Low stress → fusion
  • Mitophagy
  • The last step of mitochondrial QC pathway
  • The remaining damaged and dysfunctional mitochondria are eventually eliminated from the vital mitochondrial network
  • Mitophagy is a type of autophagy (“self-eating”) that the whole mitochondria are engulfed by autophagic membrane, leading the formation of autophagosomes
  • Works with fission and fusion
  • The mitochondria network is separated into individual mitochondria through fission. The damaged or dysfunctional individual mitochondria is then eliminated (“eaten”) through mitophagy. The remaining vital mitochondria are combined together into a new vital mitochondrial network through fusion.
  • It is highly selective process that is tightly regulated.

Mitophagy in mammalian cells is regulated via PINK 1 which is a serine/threonine kinase PTEN-induced putative kinase and the E3 ubiquitin ligase Parkin. In a healthy mitochondria PINK 1 is cleaved by PARL in the inner mitochondrial membrane. However, when there is sustained mitochondrial damage PINK 1 is able to accumulate in the outer mitochondrial membrane where it cannot be cleaved by PARL. This accumulation of PINK 1 attracts Parkin which in turn induces mitophagy.

Endosymbiont Theory edit

In 1905, Mereschkowsky, a Russian biologist, published a paper on the theory that photosynthetic bacteria are the ancestors of modern day plant chloroplasts. Though this research was mostly ignored for several years, scientists came to see the similarities between isolated living bacteria and eukaryotic mitochondria. It is now largely accepted that mitochondria are descendants of "free-living" bacteria that were engulfed and incorporated as organelles by eukaryotic cells. The endosymbiont theory was further confirmed when mitochondria were discovered to contain their own DNA. It was confirmed even more so with the discovery that the mtDNA made enzymes and proteins that were needed for its own functionalities. The fact that the mitochondria also contains a double membrane also depicts the notion that it was originally a free living organism that was later ingested into another host.[1] Mitochondria are produced by other mitochondria that act as "structural templates". A cell's two DNA genomes are still not aware of how mitochondrial membranes are assembled, showing that a mitochondria's structure isn't dictated by our DNA but must be passed on to future generations.

Mitochondria DNA edit

Mitochondria are the energy processing organelle that is found in the cell. Alongside with chloroplast, mitochondria are part endosymbiont theory, as stated above. The endosymbiont theory clearly stated the following about mitochondria and chloroplasts: it is enclosed by a double-membrane, it is about the same size as bacteria, it has its own circular DNA, its ribosomes are bacteria-like, and it has prokaryotic activities such as respiration and photosynthesis (mitochondria and chloroplasts respectively). Focusing on the third point about having its own circular DNA, mitochondria DNA (mtDNA) was found to be able to self-translate and replicate itself.

Genetics of mtDNA edit

Unlike nuclear DNA which is inherited from both mother and father, the mammalian mtDNA is only inherited from mother. The mitochondria in mammalian sperm are destroyed in the fertilized oocyte. However, the replication pathway of mtDNA is very similar to nuclear DNA. Before replication, mtDNA becomes unwind by TWINKLE, which is a protein used to undo the double-stranded DNA. After the double-stranded is undone, it is replicated at one end with the help of mtDNA polymerase. mtDNA polymerase starts to form another double-stranded starting at 5' end of the mtDNA. Another protein called mitochondrial single-stranded binding (mtSSB) helps stabilize the unwound conformation and stimulates DNA synthesis by the polymerase holoenzyme.

Unfortunately, mtDNA replication displays no strict phase specificity as in nuclear DNA synthesis. Therefore, segregation of heteroplasmic mtDNA mutation can occur as a cell divides.

Mitochondrial transcription edit

The transcription mechanism in mitochondria is likely similar to transcription in nucleus. However, there are some differences between RNA synthesis in mitochondria and in nucleus. The individual strands of the mtDNA molecules are denoted heavy strand (guanine rich) and light strand (guanine poor). This nucleotide bias explains why some codons are rare or absent in mitochondrial RNA.

The compact mammalian mtDNA genome lacks introns. The entire strand codes for either proteins, rRNA, or tRNA. Therefore, there is no need for slicing process in mitochondria

Each of the protein and rRNA genes is immediately flanked by at least one tRNA gene.

Some Interesting Discoveries about Mitochondria edit

1. Albert Claude, who was a Belgian biochemist discovered in the first half of the last century discovered that Mitochondria catalyzed respiration. He did this by isolating them through centrifugation.

2. Scientists started from there and managed to map out the flow of electrons in cellular respiration in the past two decades.

3. Peter Mitchell then discovers that the key to the flow of free energy in respiration and photosynthesis is stored within the ion gradient across membranes. He receives a Nobel Prize for it in 1978.

4. Mitochondria is bordered by 2 membranes and, and holds one tenth of the cell’s proteins. Mitochondria also converts 10,000 to 50,000 times more energy per second than the sun does.

5. Mitochondria was also discovered to play a pivotal role in programmed cell death, or apoptosis. This shows mitochondria to thus also be part of the signal transduction network in the cell. For programmed cell death, Mitochondria first releases proteins called cytochromes into the cell’s cytoplasm. It is these signals that could potentially release proteases and nucleases onto the cell and trigger cellular suicide.

6. Isolated Mitochondria were discovered to produce their own proteins even though the identity of these proteins are yet to be determined.

7. It is a theory that Mitochondria evolved from bacteria, which explains why it is such an independent organelle and doesn’t seem to depend on other parts of the cell for survival. Another evidence for this rests in the fact that the mechanism for protein synthesis in mitochondria is similar to that in bacteria.

8. Mitochondria spread by growth and division of previously existing mitochondria. Mitochondria are thus able to tell building blocks for new mitochondria where to go and what to do.

9. Recent discoveries have revealed that mitochondria actually have a lot of extramitochondrial molecules that help regulate the expression of genes that turn into mitochondrial proteins. Peroxisomal-proliferator-actived receptor coactivator 1 (PGC1) plays a major role in this process.

10. The space in between the mitochondria’s membranes was recently discovered to be able to oxidize sulfhydryl groups to disulfide bridges even though that space is surrounded by highly reducing environments.

Role in Aging edit

Scientists believe that there is a strong correlation between mitochondrial dysfunction. Mitochondrial dysfunction is one of mitochondrial diseases and is caused by reactive oxygen species (ROS). Reactive oxygen species cause oxidative damage that degrades the ability of mitochondria to make ATP. This means that mitochondria fail to carry out their metabolic functions, leading to cell death [2]. Since mitochondrial dysfunction is a factor of cell death, it is reasonable to believe that such a correlation between mitochondrial dysfunction and aging exists. It should be noted before anything that regulation of complex protein-folding environment within the organelle is vital for keeping productive metabolic output. The reason for its necessity is that without efficient metabolic output, chemical wastes and heat produced in metabolic processes, which are potential harms to the cell, cannot be transported out of the cell. Despite the fact the cells do have such complex systems to maintain efficient metabolic output, several factors come into play to prevent this. We note 2 factors here; 1. It is inevitable that over a long period of time, dysregulation of protein homeostatis arises through stress caused by the accumulation of reactive oxygen species. 2. A failure at maintaining efficient metabolic output can also be induced by mutations in the mitochondrial genome introduced during replication.

These two reasons that deteriorate mitochondria's normal functions are dependent on time; the longer a mitochondrion lives, the magnitude of these time-related effects increases. Therefore, it is believed that damage incurred on mitochondria is deeply involved in aging.

Reactive Oxygen Species (ROS) Having explained that reactive oxygen species are a crucial factor in aging, it is necessary to figure out what they really are. By definition, they are chemically reactive molecules containing oxygen. [3] These are examples of some reactive oxygen species; 1. molecules like hydrogen peroxide 2. ions like hypochlorite ion 3. radials like hydroxyl radial (this is the most reactive of all kinds of reactive oxygen species) 4. ions like superoxide anion (this is both ion and radical) [4] These reactive oxygen species are generated by the electron transport chain.

Sources of ROS in living cells

Roles of the mitochondrial proteome in aging

The mitochondrial proteome sustains the cell's cellular metabolism. Cellular metabolism inside mitochondria such as ATP production, apoptosis, and regulation of intracellular calcium. They are all essential elements to sustain life. However, the costs of maintaining such functions are the damaging effects of reactive oxygen species, as mentioned earlier. The mitochondrial proteome comprises mitochondrial and nuclear DNA-encoded proteins that needs folding and assembly within mitochondria. The two genomes that code for the structural requirements are damaged by accumulation of reactive oxygen species over time. The proteome of mammals is made up of between 1000 and 1500 proteins. Here is a summary of protein production. The list shows how proteins made in the cell are transported into mitochondria in the cell. 1. proteins are encoded in the nucleus 2. proteins are translated in the cytoplasm 3. the unfolded state of the proteins is maintained and then is imported into mitochondria. Unfolded proteins are needed to to construct ETC in mitochondria. To assist mitochondrial biogenesis and transferring of mtDNA and proteome, mitochondria must go through series of fission and fusion. Just like other organelles do, this organelle fission functions to multiply the number of mitochondria. It also serves to remove defective organelles for autophagic degradation [5]

Misfolded and misassembled mitochondrial proteins Researchers have found that inhibition of mtDNA replication, accumulation of orphaned mitochondrial complex subunits or harmful protein aggregates and ROS all can create an excessive amount of misfolded mitochondrial proteins in yeast and Caenorhabditis elegans. It should be reasonable that accumulation of such misfolded and misassembled proteins generated by those factors lead to destruction of certain mitochondrial metabolic function and its dysfunction ultimately. Here is a summary of how aging disease in mitochondria occurs 1. Reactive oxygen species accumulate inside the mitochondrion 2. This follows two possible consequences. One is that reactive oxygen species react with mtDNA and cause mtDNA mutations. It should be emphasized once again that reactive oxygen species are highly reactive agents. The other possible consequence is reactive oxygen species directly attack mitochondrial proteins. The proteins are distorted as a result. 3. mtDNA mutations caused by reactive oxygen species no longer encode for ordinary mitochondrial proteins. What encoded and translated from these mutated mtDNA are, in fact, misfolded proteins. 4. Keep in mind that proteins are used to build the complicated network of ETC. When misfold proteins are created, as long as they are present in the mitochondria, they will be used as building blocks of ETC. ETCs with misfolded proteins embedded in them no longer function properly. In other words, the mitochondria face ETC dysfunction. 5. ETCs with misfolded proteins cause to create more reactive oxygen species. As more reactive oxygen species are generated, this vicious cycle continues and misfolded proteins accumulate inside the mitochondria. Over time, the mitochondria die.

Non-native amino acids that damage the three dimensional structure of proteins are actively generated in the process of cytosolic translation of mitochondrial proteins [6]. Degrees of challenge of non-native amino acids derived from errors in cytosolic translation (under optimal conditions) - nearly 10% of newly synthesized proteins are mistranslated - 20-30 % of all nascent polypeptides are quickly denatured permanently due to folding errors

Organelle biogenesis and complex assembly Complex I of ETC is named as the NADH-ubiquinone oxidoreductase. Complex I is known to possess about 45 subunits. And mutations or functional failures are known to be potential causes of neurodegenerative diseases. Such diseases include Parkinson's disease. Out of the 45 subunits of ETC, 7 of them are encoded by the genome in mitochondria. They need to be embedded into the mitochondrial inner membrane because that is where they build stoichiometric complexes with nuclear-encoded components. Suppose that one of the subunits was misexpressed due to mutation. Then the entire network of ETC is doomed to collapse. In other words, mutations or deletions of ETC's subunits (even just single mutation or deletion), have a tremendous effect on whole complex formation. This illustrates the significance of the coordination of genome of proper complex assembly and function.

Mitochondrial compartments There are four compartments in mitochondria where protein folding and assembly happens. The four compartments are the outer membrane, intermembrane space, inner membrane and matrix [7]. It should be stressed that misfolded proteins can build up in those compartments. The interesting fact is that there exists a structure in the mitochondria that monitors these levels of accumulation. Compartment-specific QC machinery is the one which is responsible for monitoring the accumulating unfolded proteins. Normal fold proteins have hydrophobic amino acids buried inside them and their heads often stick out. Caperones and QC proteases then come into place to acknowledge these hydrophobic amino acid heads. We should divide how the QC monitors the level of unfolded proteins into each compartment. Outer Membrane It seems from recent evidence that cytosolic chaperones and the ubiquitin-proteasome system are involved in the mechanisms that regulated quality control of mitochondrial protein import or outer membrane proteins. Nuclear-encoded proteins which are ordered to form mitochondria are translated in the cytoplasm. The chaperones are used to sustain precursor proteins that are unfolded or misfolded. They also serve to prevent protein accumulation during delivery to the translocase of the outer membrane channel

Damage done by different types of reactive oxygen species

-reactive oxygen species attack mitochondrial proteins and DNA during cell division -superoxide anion is derived from dominantly from complexes I and III of the ETC during oxidative phosphorylaiton. Superoxide anion, having a strong negative charge upon it, can deteriorate all four mitochondrial compartments. - storing up an excessive amount of reactive oxygen species could totally overwhelm mitochondrial ROS-detoxifying systems. - reactive oxygen species may directly interfere with protein folding by changing its amino acid sequence, giving its secondary and tertiary structures inevitable and irreversible changes - reactive oxygen species may indirectly interfere with protein folding by introducing mutations in genes encoded by mitochondrial or nuclear DNA. - note that mtDNA is very vulnerable to oxidative damage because it is located ner the site of reactive oxygen species production and does not have histone protection

Age-associated organelles damage In the long run, the decrease in mitochondrial function caused by reactive oxygen species and mtDNA and others leads to the onset of progressive age-associated pathologies [8] 1. cancer 2. neurodegeneratin 3. hearing loss It is should be aware that accumulation of destruction caused by free radical is one of the most commonly cited effects of age-related mitochondrial dysfunction.

Researchers have used knockin mouse strains that express error-prone mtDNA polymerases that ignore proof-reading activity. The mous matures normally, but showed properties that are determined to be accelerated aging. These symptoms included; - apoptosis - curvature of the spine - reduced fertility - weight loss - early death [9].

Recent research into aging has lead scientists to believe that damage done by free radical oxygen may be one reason why organisms die from old age. Reactive Oxygen Species, or ROS, are produced in greatest quantity at the mitochondria, so this organelle is the most likely to be damaged by the free radical oxygen. Increases in ROS damage the mitochondria in several ways. They can modify amino acids and mutate the genes in mitochondrial and nuclear DNA, all affecting proper protein folding. Furthermore, ROS causes oxidative damage to mitochondrial DNA (mtDNA), especially because mtDNA is near the ROS production area[10]. Mutations on the mtDNA could result in reduced ATP production, increased ROS production, and eventual apoptosis. The increased ROS production has the added effect of being potentially harmful to the cell hosting the mitochondria, which could cause mutation in the cell DNA.

Anti-aging research has shown in some model organisms that by genetically disrupting the function of mitochondria life span has been increased. This is because ROS production was decreased so that less damage was done to the mtDNA. Specifically, a reduction in the mitochondrial function of the electron transport chain (ETC) in c. elegans increased longevity of the organism.

Scientists hope to be able to apply this to extending human lifespan by somehow incorporating reduced mitochondrial function with a treatment of dietary restriction (DR). By reducing the amount of calories taken in, but not to the point of starvation, cellular processes would change such that emphasis is placed on maintaining existing cellular structures rather than generating new structures to replace old structures. This would cause cells to persist in the body longer and there would be fewer mutations due to gene replication during mitosis.

Recent studies have underscored the importance of mitochondrial quality control (QC) pathways such as chaperones and proteases in solving mitochondrial dysfunctions. The protease complex "Lon" contains a cofactor made of iron-sulfur clusters; it is therefore suspected that Lon plays a role in targeting proteins by making them susceptible to oxidative damage. Findings suggest that a lack of Lon in yeast and mammalian cells results in excess proteins and mtDNA deletions, as well as respiration loss[11]. These all suggest that Lon plays a role in quality control systems in the mitochondria. On the other hand, over-expression of Lon in the fungus Podospora anserina leads to greater cell life span[12]. This suggests that there can be ways to artificially enhance the activity of proteases in order to increase cell longevity. Although many studies have strongly implied the importance of chaperones and proteases in anti-aging properties, it is important to note that fewer studies have been done in mammals.

Mitochondrial protein import edit

Although mitochondria have unique genetic and protein synthesis system, majority of mitochondrial proteins are synthesized as precursors in the cytosol and are imported to mitochondria from the cytosol. Now, five different protein import pathways have been distinguished. Since these pathways cooperate with each other and are connected to other systems that function in the respiratory chain, mitochondrial membrane organization, protein quality control and endoplasmic reticulum-mitochondria junctions, mitochondrial protein import is highly responsible for major mitochondrial functions.

Two classical import routes:

Classical import routes include presequence pathway and carrier pathway. Both pathways use the core of TOM (translocase of the outer mitochondrial membrane) complex, which is the main protein entry gate of mitochondria including Tom 40, to transport precursor from the cytosol to intermembrane space.

Presequence pathway targets proteins carry cleavable presequences, which are peptide extensions of about 10 to 60 amino acids residues located at the N-terminal end of the protein

  1. Cleavable precursor proteins are recognized by the receptor Tom20 and Tom22 and are transported across the outer membrane from the cytosol to intermembrane space through Tom40 channel
  2. Cleavable precursor proteins are then transferred to Tim23 complex (presequence translocase of the inner mitochondrial membrane) with the help of intermembrane space-exposed proteins, including Tim25 and Tim22. The membrane potential of the inner membrane enables Tim23 channel to translocate the preseqyebces across the inner membrane. The transport of protein into the matrix requires the ATP that powers the PAM (presequence translocase-associated motor).
  3. Presequences-carrying precursors are inserted into the inner membrane by two different ways. A) Many presequence-containing inner membrane proteins are laterally released from the Tim23 complex as the inner membrane proteins. Some of the hydrophobic sorting signal of some presequences are removed by the inner membrane peptidase complex and released into the intermembrane space as the intermembrane space proteins. B)Other precursor proteins are first transported into the matrix and eventually integrated into the inner membrane through Oxa1 export complex.

Carrier pathway transports non-cleavable precursor proteins into inner membrane.

  1. Non-cleavable precursors are recognized by the receptor Tom70 and are transported across the outer membrane from the cytosol to intermembrane space through Tom40 channel
  2. The precursor proteins are then guided by small TIM chaperone, such as Tim9-Tim10 complex, to the Tim22 complex (the carrier translocase of the inner mitochondrial membrane). The membrane potential of the inner membrane enables Tim22 to insert precursor proteins into the inner membrane as the carrier proteins.

Two routes of inserting proteins into the outer membrane:

The outer membrane of mitochondria contains two classes protein: β-barrel proteins, which are probably derived from the bacterial ancestor of mitochondria, and proteins with α-helical transmembrane segments, which are probably derived from the eukaryotic cell. The two routes of inserting two different proteins are called β-barrel pathway (SAM) and α-helical insertion (Mim1) reprehensively. Although SAM and Mim1 complexes insert precursor proteins independently, they can cooperate with each other to assemble some outer membrane complexes, such as TOM complex, which consists of central beta-barrel protein Tom40 and some α-helical subunits.

β-barrel pathway inserts β-barrel protein via SAM complexes

  1. β-barrel precursors are recognized by TOM receptors and are translocated to the intermembrane space by Tom40 channel.
  2. β-barrel precursors are then guided by small TIM chaperone to the SAM complex, Sam50 and Sam35, and are converted to outer membrane β-barrel proteins, which is also linked to ER-mitochondria junctions due to the dual localization of Mdm10 in SAM and ERMES, ER-mitochondria encounter structure

α-helical insertion inserts most Tom proteins, which have a single α-helical transmembrane segment, and outer membrane proteins with multiple α-helical transmembrane segments via Mim1, which contains one α-helical transmembrane segment.

  1. α-helical precursors are recognized by Tom and are transferred to Mim1.
  2. α-helical precursors are then interacted with Mim1 and are inserted into the outer membrane. [The exact function of Mim1 and how Mim1 really inserts precursor proteins is still unknown]

Protein import into the intermembrane space:

Mitochondrial intermembrane space assembly pathway (MIA) targets the intermembrane space precursors, which are cysteine-rich.

  1. The precursors are transferred from cytosol to intermembrane space though TOM complexes in a reduced state.
  2. When the precursors are in the intermembrane space, the precursors are oxidized and are immediately bound with Mia40, forming a transient disulfide bond. Then, Mia40 forms the oxidative folding of proteins, Erv1, to function as disulfide relay. Erv1 catalyzed the formation of disulfide bonds in Mia40 that oxidizes cysteines in the precursor proteins. As a result, new disulfide bonds are formed. Electrons flow from Mia40 to Erv1 and eventually to respiratory chain. In other words, Mia40 acts as a receptors that recognizes the precursors and enables the translocation of precursors into intermembrane space.

References edit

  1. The Evolution of the Cell
  5. “Mitochondrial protein quality control during biogenesis and aging” by Brooke M. Baker, Cole M. Haynes
  6. “Mitochondrial protein quality control during biogenesis and aging” by Brooke M. Baker, Cole M. Haynes
  7. “Mitochondrial protein quality control during biogenesis and aging” by Brooke M. Baker, Cole M. Haynes
  8. “Mitochondrial protein quality control during biogenesis and aging” by Brooke M. Baker, Cole M. Haynes
  9. “Mitochondrial protein quality control during biogenesis and aging” by Brooke M. Baker, Cole M. Haynes.
  10. Brooke M. Baker, Cole M. Haynes, Mitochondrial protein quality control during biogenesis and aging, Trends in Biochemical Sciences, Volume 36, Issue 5, May 2011, Page 255, ISSN 0968-0004, 10.1016/j.tibs.2011.01.004. <>
  11. Brooke M. Baker, Cole M. Haynes, Mitochondrial protein quality control during biogenesis and aging, Trends in Biochemical Sciences, Volume 36, Issue 5, May 2011, Page 258, ISSN 0968-0004, 10.1016/j.tibs.21.01.00014. <>
  12. Brooke M. Baker, Cole M. Haynes, Mitochondrial protein quality control during biogenesis and aging, Trends in Biochemical Sciences, Volume 36, Issue 5, May 2011, Page 259, ISSN 0968-0004, 10.1016/j.tibs.2011.01.004. <>

IF1: setting the pace of the F1Fo-ATP synthase. Campanella, M. Trends in Biochemical Sciences, Volume 34, Issue 7, 343-350, 25 June 2009.

Scherz-Shouval, Ruth, et al. Regulation of autophagy by ROS: physiology and pathology

Schatz, Gottfried. The Magic Garden, Annual Review of Biochemistry, 2007: 673-78.

Falkenberg, M., Larsson, N., & Gustafsson, C. M. (2007). DNA replication and transcription in mammalian mitochondria. Annual Review of Biochemistry, 679-699.

Mair, William, and Andrew Dillin. "Aging and Survival: The Genetics of Life Span Extension by Dietary Restriction." Annual Review of Biochemistry 77.1 (2008): 727-54. Web

Wager, Peter. Exercise Physiology. Aug 17,2011.

Becker, Thomas, Lena Böttinger, and Nikolaus Pfanner. "Mitochondrial Protein Import: From Transport Pathways to an Integrated Network." Trends in Biochemical Sciences 37.3 (2012): 85-91.

Hamanka, Robert B., and Navedeep S. Chandel. "MItochondrial reactive oxygen species regulate cellular signaling and dictate biological outcomes." Trends in Biochemical Sciences 35 (2010): 505-13.

Fischer, Fabian, Andrea Hamann, and Heinz D. Osiewacz. "Mitochondrial quality control: An integrated network of pathways." Trends in Biochemical Sciences 37 (2012): 284-92.

Slonczewski, Joan L. Microbiology "An Evolving Science." Second Edition

Overview edit


Cardiolipin is known as the signature phospholipid of mitochondria. It is responsible for a wide range of mitochondrial functions, including but not limited to ATP synthesis. Therefore, disorientation and disturbance in the metabolism of cardiolipin can cause pathological issues. Recently, there is a study on the enzymes that participate in cardiolipin biosynthesis and remodeling. After biosynthesis of cardiolipin, there are three remodeling enzymes that modify the acyl chain composition of cardiolipin, producing either a “dysfunctional” cardiolipin that is tied to michondrial dysfunction or a tissue-specific cardiolipin in its mature form. The following will discuss newly found molecules involved in cardiolipin metabolism as well as how human diseases are affected by the changes in cardiolipin metabolism.

Cardiolipin Background edit

Cardiolipin formation is key to the enzyme functionality of the mitochondria thereby with it malfunctioning mitochondria related diseases can occur. This compound is referred to as the “signature phospholipid” of the mitochondria because it is always present as well as is being formed there. It also appears to promote ATP formation which is the major role of the mitochondria.

Figure 1 Cardiolipin Structure

The Phospholipid Uniquely in Mitochondria edit

Cardiolipin improves the efficiency of the OXPHOS machinery.

When cardiolipin is present in an organelle’s membrane, that organelle must be a mitochondrion. This is the reputation of cardiolipin, the signature phospholipid of mitochondria. There, there is no surprise that most of the cardiolipin in a cell is closely tied to the inner membrane of mitochondria. Furthermore, the cardiolipin synthesis actually takes place in the inner membrane of mitochondria. Unlike other phospholipids that are also synthesized in a special compartment of the cell, cardiolipin remains closely associated with the mitochondrial membranes and does not get distributed out to the other endomembrane system like other phospholipids do. Because of this close association between cardiolipin and mitochondria, it is hypothesized that cardiolipin plays a huge role in ATP production through oxidative phosphorylation. In addition to this, since cardiolipin is only found in membranes that has an electrochemical gradient, it therefore also supports that cardiolipin has something to do with ATP production in the powerhouse of the cell. In addition to oxidation phosphorylation, cardiolipin also play a role in many mitochondrial activities. Cardiolipin contains two phosphatidyl groups connected by a glycerol. For this reason, cardiolipin is a lipid dimer that has four acyl chains instead of two as seen in other phospholipids. Out of all the biosynthetic enzymes except for cardiolipin synthase, they do not have any acyl chain specificity. Thus, the last acyl chain composition is not made during biosynthesis but rather in deacylation-reacylation/transacylation reactions. When added together, these reactions make the final form of cardiolipin cell/tissue-specific. In other words, different organisms can have different final molecular forms of cardiolipin and even in different tissues in the same organism. This proposes that the different forms of cardiolipin exist for the different tissues and cells function, mainly to accommodate the functions and energetic demands of the different tissues/cells. However, we are still unsure of whether having different acyl chain compositions will affect the function itself. We do know that the Barth syndrome is associated with the first inborn error of cardiolipin metabolism, since its cardiolipin is alternated in biosynthesis. So far, there are three known cardiolipin remodeling pathways as well as known proteins in the different stages of the cardiolipin biosynthetic pathways.

Recently found biosynthetic pathways edit

Cardiolipin biosynthesis takes place in the mitochondrion of eukaryotic cells. The enzymes of the dephosphorylation of phosphatidylglycerolphosphate and initiation of the cardiolipin remodeling cascade were recently found.

Phosphatidylglycerolphosphate phosphatase

Matrix IM protein import

The proteins Gep4 and PTPMT1 were identified in the phosphatidylglycerolphosphate phosphatases. Initially, Gep4 was detected in a screen in yeast as genetic interactors of prohibitions, which led to identifying Gep4 as a key player in the phosphatidylglycerolphosphate phosphatase of cardiolipin synthetic pathways. Indeed, a gep4 null has small amounts of cardiolipin and phosphatidylglycerol, which build up phosphatidylglycerolphosphate and destabilized respiratory supercomplexes. Furthermore, this is not able to grow on carbon sources that need a working OXPHOS system. In addition, recombinant Gep4 dephosphorylates phosphatidylglycerolphosphate to phosphatidylglycerol in experiments conducted in the laboratory.

Recently, the function of the substrates of PTPMT1 was discovered. PTPMT1 is part of the phosphatase and tensin homolog family that lives in the mitochondria. Knockout mices of PTPMT1 died in the uterus before embryonic day 8.5, showing that PTMPT1 is necessary for life. In observing the mouse embryonic fibroblasts of the PTPMT1 knockouts, slow growth, OXPHOS defects, reduced complex I levels, and altered inner membrane morphology were all seen. In addition, cardiolipin and phosphatidylglycerol levels decreased while phosphatidylglycerolphosphate accumulated. Like Gep4, recombinant PTPMT1 dephosphorylates phosphatidylglycerolphosphate to phosphatidylglycerol. While Gep4 is only present in plants and fungi, PTPMT1 is conserved evolutionarily. Still, these two proteins play a major role in the inner membrane of mitochondria and catalyze the same reaction. Interestingly enough, PTPMT1 is able to rescue a Geo4 null. Even though cardiolipin is present on both sides of the inner membrane, the two proteins Gep4 and PTPMT1 reside in the matrix face of the inner membrane only.

Cardiolipin phospholipases

The removal of a single acyl chain leads to monolysocardiolipin, which initiates cardiolipin remodeling. In yeasts, tafazzin (Taz1) is the transacylase that participates in cardiolipin remodeling. It does so by taking an acyl chain from another phospholipid and connecting it to the monolysocardiolipin. In the upstream of Taz1, cardiolipin deacylase 1 (Cld1) resides there. Surprisingly, Δcld Δtaz1 yeasts does not have monolysocardiolipin and has normal level of cardiolipin. However, Cld1 is observed to be involved in more than one metabolic pathways. This is due to the growth phenotype of the Δcld1 Δtaz1 strain is more disastrous than just one mutant. Secondly, Cld1 can add water to phospholipids as well as cardiolipin.

Formation Cardiolipin and Functionality edit

This molecule proceeds through a maturation process before becoming functional following the steps:

Figure 2 Simplification of the Cardiolipin Maturation Process in the Mitochondria

The pathway for forming Cardiolipin requires many enzymes, 1 for each step. Problems with Cardiolipin formation may result in lowered functionality of the mitochondria implying the following roles of the Cardiolipin in the mitochondria: Increasing efficiency of the OXPHOS- oxidative phosphorylation involved in ATP formation

  • Stabilized assemblies involved in this process
  • Traps protons resulting in changes in the gradient which is involved in the process that produces the most ATP in the cycle which results in an increasing gradient and a in turn a greater ATP production
  • Help in the process of cell death
  • Is key to other compounds being able to recognize and identify the mitochondria

An important aspect to proper activation of the Cardiolipin are the remodeling process of which there are three without which it does not function properly. There are three key enzymes that promote this remodeling process:

  • Tafazzin
  • Monolysocardiolipin acyltransferase 1
  • Acyl-CoA: lysocardiolipin acyltransferase 1

The major diseases involved in the malfunctioning of these include: Wilson’s disease and Barth syndrome.

Conclusion edit

While the studies in cardioipin metabolism and how it can lead to human diseases is still new, there is a consistent effort to conduct experiments. So far, we have the complete biosynthetic pathway and remodeling inventory. For future steps, the regulation of cardiolipin metabolism and how it relates to health can be further studied. With the increasing interest and models in search of answers, we are close to accumulating more knowledge for this signature phospholipid of mitochondria.

References edit

Claypool, Steven M., and Carla M. Koehler. "The Complexity of Cardiolipin in Health and Disease." Trends in Biochemical Sciences 37.1 (2012): 32-40. PubMed. Web. 3 Dec. 2012.

Zhang, J; Dixon JE (6-8-2011). "Mitochondrial phosphatase PTPMT1 is essential for cardiolipin biosynthesis". Cell Metab 13 (6): 690–700. doi:10.1016/j.cmet.2011.04.007. PMID 21641550.

Overview edit

3D view of a centriole

Every animal cell has two small organelles called centrioles. Centrioles help the cell when it comes time to divide. Mitosis and meiosis both have centrioles involved in. A centriole is a small set of microtubules arranged in a specific way. Also, there are nine groups of microtubules. The centrioles are found in pairs and move towards the poles of the nucleus when it is time for cell division. During interphase, the cell is at rest. The centrioles move to opposite ends of the nucleus and a mitotic spindle of threads begins to appear during prophase.

Centrioles are cylindrical structures that composed of groupings of microtubules arranged in 9+3 pattern. They are usually exist in pairs that form centrosomes. Centrioles are found in animal cells and play a big role in cell division. While cell division, the centrosome divides and the centrioles replicate (make new copies). The result is two centrosomes, each with its own pair of centrioles. The two centrosomes move to opposite ends of the nucleus, and from each centrosome, microtubules grow into a "spindle" which is responsible for separating replicated chromosomes into the two daughter cells. Centrioles contain delta-tubulin, a protein which is part of the structure of tubulin. Centrioles are barrel-shaped organelles that are found in most eukaryotic cells, with the exception of vascular plants and fungi. Centrioles consist of nine triplets of microtubules and are involved in the mitotic spindling, as well as when the cytoplasm of a eukaryotic cell split to form two daughter cells. The intracellular fluid inside a cell is called the cytosol. It is separate from certain cell organelles such as the nucleus and the mitochondria. In eukaryotes, the cytoplasm is the content within a cell membrane minus the content in the cell nucleus. It is where many metabolic reactions occur, there are still others within organelles. In prokaryotes, cytosol is where most metabolic chemical reactions occur. These reactions also occur in the membranes or in the periplasmic space but to a lesser extent. A complex mixture of substances dissolved in water forms the cytosol even though water is the large majority of the mixture. Sodium and potassium concentrations are different in the cytosol compared with the concentrations in the extracellular fluid. The difference in ion levels are important for osmoregulation and cell signaling. Besides ions, the cytosol also has macromolecules.

Cytoplasm vs. Cytosol edit

There is often much confusion between the cytoplasm and the cytosol. The cytoplasm is the fluid contained within the cell that holds and surround the cell's organelles in a liquid environment which is necessary for many of the cell's vital functions to occur. Some of the organelles that are held within the cytoplasm include the mitochondria, the golgi apparatus, the endoplasmic reticulum, and other organelles. One thing to note is that the nucleus isn't considered to be part of the cytoplasm because it contains its own type of fluid-like material that is referred to as the nucleoplasm. The materials that are found within the cytoplasm are also typically found within the cell membrane. Typically, the cytoplasm contains materials that are known as cytoplasmic inclusions. These inclusions are typically starch granules, mineral crystals, or lipid droplets that are floating around within the cytoplasm.

When looking at the two, the cytosol is often confused as being the cytoplasm itself, and many individuals view the two as being synonymous. However, they cytosol is in fact just a part of the cytoplasm. The gel-like translucent fluid is what the cytosol really is. It is typically not enclosed within the organelles found within the cell. The cytosol is responsible for suspending the other elements contained within the cytoplasm, like the cytoplasmic inclusions and the cell organelles. Consisting of around 75% of the cell's total volume, the cytosol is composed of many different cellular components. Concentration gradients, cytoskeletal sieves, protein complexes, protein compartments, water, salts, organic compounds, and dissolved ions all consist of the contents of cytosol. Structurally, the cytosol consists mostly of water. However, it does house cytoskeleton filaments, which are necessary for maintaining the overall structure of the cell. In regards to function, the cytosol is also the location where many of the cell's chemical reactions occur. Transportation of metabolite and cell communication are among some of the important functions that occur within the cytosol.

Cytosol and Importing tRNA into Mitochondria

It has been observed that the mitochondria compensate for its lack of mitochondrial tRNA genes by taking in tRNA contained within the cytosol of the cell. The organelle does this by co-importing tRNA along with mitochondrial precursor proteins that intercepts the organelles pathway for traditional protein importing. This method also illustrates the complexity of mitochondrial protein importing, because specific modifications and mitochondrial precursor proteins are required to carry out imports for certain proteins; and with the variety of proteins that the organelle imports, this method possesses a unique set of problems. Another, more direct method, of obtaining more tRNA can be directly importing the tRNA without the help of mitochondrial precursors; a method that is seen in even earlier protozoa.


Commonly seen, protein molecules that do not bind to cytoskeleton or cell membranes are simply because they dissolve in the cytosol. Since the amount of protein in cells approaches up to 200 mg/ml, it is known to be very high. Protein also occupies approximately 20-30% of the volume of cytosol. However, even with this many protein occupying cytosol, some proteins with the membranes or organelles in cells are known to be very weak and are eventually released into solution upon cell lysis.

If given ATP and amino acids, cells are able to synthesize proteins. This shows that many of the enzymes in cytosol are bound to the cytoskeleton in the cell. In cells such as prokaryotes cell, the cytosol within nucleoid contains the cell's genome. This is known to be an unusual mass of DNA and associated proteins that control the replication and transcription of the bacterial chromosomes and plasmids.

On the other hand, cells such as eukaryotes cell the cytosol containing the cell's genome is held within the cell nucleus, which then separates from the cytosol by nuclear pores that blocks the free diffusion of any kind of molecule that is larger than 10 nm in diameter.

The effect of macromolecular crowding is cause because of too high concentration of macromolecules in the cytosol. In other words, this happens when the effective concentration of other macromolecules is increased, since they have less volume to move in. This squishy effect can cause the position and rate of chemical equilibrium of the cytosol's reactions. Sometimes in the genome forming protein complexes or DNA binding proteins occurs to the favor the association of marcromolecules.

References edit

Schneider A., “Mitochondrial tRNA import and its consequences for mitochondrial translation.” Annu Rev Biochem. 2011 Jun 7;80:1033-53. Print

Lysosome edit

Lysosomes are spherical bodies, or vacuoles that are enclosed by a single membrane. The membrane serves as a protectorate to the cell, since lysosomes contain harsh digestive enzymes, which would cause significant damage if exposed to cell content. Lysosomes contain different hydrolytic enzymes, such as proteases, lipases, and nucleases that are capable of breaking down all types of biological polymers (e.g. proteins, nucleic acids, carbohydrates, and lipids) that enter the cell or are no longer useful to the cell. In all, lysosomes function as the digestive system of the cell. On another note, lysosomes avoid self-digestion by glycosylation of inner membrane proteins, which prevent their degradation.

Structure of Lysosome

Lysosomes are membrane-enclosed organelles that help eukaryotic cells obtain nourishment from macromolecular nutrients. The lysosomes contain many hydrolytic enzymes such as proteases, nucleases, and lipases). The lysosomes are formed vesicles containing hydrolytic enzymes and proton pumps bud off from the Golgi complex. Phagocytosis and lysosomal digestion help the eukaryotic cell because they effectively increase the membrane surface area over which nutrients can be absorbed. However, in eukaryotes, lysosomes allow for intracellular digestion, and digested material crosses the lysosomal membrane into the cytoplasm.

Structure of Lysosome

Lysosome Production edit

Lysosomes are manufactured and budded into the cytoplasm by the Golgi apparatus with enzymes inside. The enzymes that are within the lysosome are made in the rough endoplasmic reticulum, which are then delivered to the Golgi apparatus via transport vesicles. When lysosome reaches the cytoplasm, fusion forms a secondary lysosome.

Structure of Lysosome

Inside Lysosomes edit

Lysosomes contain enzymes that include:

  • Glycosidase: an enzyme that catalyzes the hydrolysis of glycosidic linkages in sugar molecules.
  • Protease: an enzyme that catalyzes the hydrolysis of peptide bonds.
  • Acid phosphatase: a phosphatase, an enzyme that catalyzes they hydrolysis of phosphate linkages in a variety of molecules.
  • Sulfatase: an enzyme that catalyzes the hydrolysis of sulfate ester linkages in molecules.
  • Lipase: an enzyme that catalyzes the hydrolysis of ester bonds in lipid molecules.
  • Amylase: an enzyme that catalyzes the breakdown of carbohydrates into sugars.
  • Nuclease: an enzyme that catalyzes the hydrolysis of phosphodiester bonds in nucleotide subunits of nucleic acids.
    Structure of Lysosome
  • Glucocerebrosidase: an enzyme which breaks down glucocerebroside
  • Phosphoric Acid monoesters

Lysosome enzymes are proteins that are synthesized in the endoplasmic reticulum and modified in the Golgi apparatus. Lysosomal enzymes are tagged for lysosomes by the addition of mannose-6-phopahte label. A shortage of any one of these enzymes will lead to lysosome diseases such as Tay-Sachs disease and Pompe’s disease.

Lysosomal Defects edit

In human cells, when lysosomes lack enzymes they can generate storage disease

Structure of Lysosome

. People with these defects are usually missing one or more of the Lysosomal hydrolysis enzymes.

Tay-Sachs Disease
Tay-Sachs disease is a disease that affects the nervous system. It occurs when the body lacks hexosaminidase A, a protein that breaks down ganglioside. Accumulation of ganglioside
Pompe’s disease
Pompe’s disease is a disease that affects the heart
Structure of Lysosome
and skeletal muscles. It occurs when the body lacks alpha-glucosidase, an enzyme that breaks down glycogen.
Mucopolysaccharidosis I
MPS I is cause by the deficiency of the lysosomal enzyme alpha-L-iduronidase. Deficiency can effect both skeletal and mental development.
Gaucher’s disease
Gaucher’s disease is caused by the deficiency of the lysosomal enzyme glucocerebrosidase enzyme. A deficiency of this enzyme causes harmful substances to build up in liver, spleen, bones, and bone marrow.

Lysosomal Enzyme Production edit

Enzymes that are used by the lysosome are synthesized in the endoplasmic reticulum (ER) and are transported to the Golgi apparatus (GA). The ER formed transport vesicles that carry the enzymes and loads them by fusing them to the GA. Glycosylation may occur, which will determine the final destination of the proteins.

Lysosome Acid-Hydrolyses edit

Lysosomes contain a variation of enzymes that hydrolyze proteins, nucleic acids, carbohydrates, and lipids. The enzymes present in the lysosomes work best at an acidic pH; thus they are referred to as acid hydrolases. Is it evident to note that the pH in a lysosome is about ~5 while the cytoplasm has a neutral pH of about ~7. In order for the lysosome to maintain an acidic pH, the lysosome must actively uptake concentrate H+ ions (protons). The concentrated H+ ions are actively equilibrated to reach a pH of about 5 by hydrogen ion pumps. In part with the pH level, enzymes within a lysosomes work best at an acidic pH.

Function edit

Lysosomes digest unused and/or threatening content that is present in the cell. In retrospect, lysosomes recycle the cell’s organic material in a process known as autophagy. It also recycles waste products such as fats, carbohydrates, proteins, and other macromolecules into simple compounds, which are then utilized as new materials in the cytoplasm. The recycling of waste product is in large part due to about 40 different types of hydrolytic enzymes that are engineered by the endoplasmic reticulum and modified by the Golgi apparatus.

Autophagy (Autophagocytosis)
The term autophagy is derived from the Greek “auto” or oneself and “phagy” to eat. Autophagy is a process of self-eating and self-degradation. The degradation takes part when the cell content and organelles is consumed by lysosomes. The cell produces vesicles called autophagosomes that captures and deliver cytoplasmic material to lysosomes. Autophagosomes are formed when the ER wraps around damaged cell structures or cell content, of which the lysosome then joining the autophagosomes to destroy the content captured and delivers the broken down material to the cytoplasm. Overall, autophagocytosis preserves the health of cells and tissues by degrading unused and/or damaged cellular content to new ones.
Endocytosis occurs when the cell membrane of a cell surrounds large nutritional molecules. This engulfing of a large nutritional molecule forms a endosome. Some of the things endosomes acquire are complex lipids, polysaccharides, nucleic acids, and proteins. Lysosomes then fuse with endosomes, whereby it transfers it enzymes to breakdown molecules. The broken down molecules are then delivered to the cytoplasm by proteins for later consumption.
Lysosome works in close relations with phagocytosis whereby it joins phagosomes to break down the object or bacteria with its enzymes. Phagosomes are produced when the cell membrane of a cell surrounds a disease-causing bacterium; toxic or unused content in/outside the cell.

Background Information edit

Cell Structure. Letter i corresponds to the Peroxisome

They are theorized to have evolved from bacteria that formed a symbiotic relationship with their host cell. It was believed that the development of this relationship over generations led to bacteria evolving as an organelle inside the body. This view has recently been countered since it was found that cells without peroxisomes can restore these peroxisomes with a simple gene introduction. Therefore, the previous theory was challenged since the introduction of peroxisomes is not an evolutionary process and can easily be replicated.

Peroxisome Structure edit

Analyzing the structures of peroxisomes has helped figure their function and role in the biological world. Peroxisomes are not derived from the endoplasmic reticulum and therefore are not a part of endomembrane system. They replicate by fission. This organelle is surrounded by a lipid bilayer membrane which encloses the crystalloid core. The bilayer is a plasma membrane which regulates what enters and exits the peroxisome. There are at least 32 known peroxisomal proteins, called peroxins, which carry out peroxisomal function inside the organelle.

Peroxisome Structure

Peroxisome Function edit

The main function of peroxisomes is to break down long fatty acid chains through beta-oxidation and synthesize necessary phospholipids (such as plasmologen) that are critical for proper brain and lung function. Furthermore, they aid certain enzymes with energy metabolism in many eukaryotic cells as well with cholesterol synthesis in animals. Peroxisomes are also involved in germinating seeds in the glyoxylate cycle, photosynthesis in leaves, and oxidation of amines in various yeasts.

During catabolism of fatty acid chains in animal cells, peroxisomes break down long fatty acids into medium fatty acids which are then transported to mitochondria where the majority of catabolism happens. However, in yeast and plant cells, catabolism of fatty acid chains happens only in the peroxisome and the mitochondria is not involved. The catabolism of fats and fat-soluble vitamins, such as vitamin A and vitamin K, as well as the production of bile acids also takes place in peroxisomes. If this catabolism is not done properly, genetic disorders or skin disorders often result.

The synthesis of plasmalogens in animal cells also takes place in peroxisomes. These organelles are very important to the cell because production of plasmalogens is critical to proper functioning of the nervous system since a lack of plasmalogens causes abnormalities in the myelination of nerve cells.

Peroxisomal Disorders edit

Peroxisomal disorders may result due to abnormalities in single enzymes or groups of proteins that are necessary for normal peroxisome function. These disorders can affect a range of organ systems, but problems with the nervous system are the most commonly observed. Along with brain damage, many of these disorders also lead to skeletal and craniofacial dysmorphism, liver dysfunction, progressive sensorineural hearing loss, and retinopathy. Some examples of peroxisomal disorders:
1. Adrenoleukodystrophy: a fatal inherited X-linked disorder that leads to extensive brain damage and adrenal gland failure.
2. Zellweger syndrome spectrum (PBD-ZSD): a rare cerebrohepatorenal syndrome evolving due to a lack of functional peroxisomes.
3. Rhizomelic chondrodysplasia punctata type 1 (RCDP1): a rare brain disorder evolving due to shortening of the proximal bones.

Peroxisomal Evolution edit

Due to peroxisome's ability to "divide and import proteins post-translationally," it is suggested that it is similar to the mitochondria where an endosymbiotic relationship was formed in its origin.

Studies have shown that: (39–58%) of peroxisome are of eukaryotic origin (13–18%) are enzymes from the mitochondria

This lead to the conclusion that it did not have a endosymbiotic origin, but rather it used proteins from other eukaryotic cells.


References edit

  1. 1. Wanders RJ, Waterham HR. (2006). Biochemistry of mammalian peroxisomes revisited.'. "PubMed"
    2. (2011). Peroxisome.'. "Wikipedia"
    4. Alberts, Johnson, Lewis, Raff, Roberts, Peter Walter (2002). Peroxisomes.'. "National Center for Biotechnology Information"

The structural biochemistry of the cytoskeleton is very essential to the cell body

Overview edit

The cytoskeleton provides support in a cell. It is a network of protein fibers supporting cell shape and anchoring organelles within the cell. The three main structural components of the cytoskeleton are microtubules (formed by tubulins) , microfilaments (formed by actins) and intermediate filaments. All three components interact with each other non-covalently. Eukaryotic cells contain proteins called intermediate filaments, microfilaments, and microtubules that are collectively termed the cytoskeleton. Also, the cytoskeleton proteins are multifunctional and are also involved in whole-cell movements and movements of substances within the cell.

Actins and Tubulins are abundant cytoskeletal proteins which support diverse cellular processes due to their unique properties of filament-forming proteins. Recent evidences suggest that regulated degradation pathways exist for actin and tubulin collectively maintain the quality control of cytoskeletal proteins, ensuring the appropriate function of microfilaments and microtubules.[1]

Microtubules edit

Microtubules are polymers of tubulin. They help with cell transport. They also help with the cell shape because it resists compression. It also helps facilitate cell motility. There are two motor proteins that assist organelles to move along the microtubules:

  • Kinesin, which moves things away from the nucleus.
  • Dynein, which moves things towards the nucleus.

Microtubles have a larger diameter than microfilaments and intermediate filaments. The hollow microtubule structure consists of 13 tubulin dimers. They are one alpha-tubulin protein plus one beta-tubulin protein form one tubulin dimer. Microtubules also help movement of substances within the cell and are also involved in powering whole-cell movement by cilia and flagella. The microtubules provide tracks that can move vesicles from one organelle to the next in an efficient, directed fashion. Microtubules also segregate the duplicated chromosomes during mitosis.

Microfilaments edit

Microfilaments are polymers of actin. They help with cell shape also because it bears tension in the cell. It is also involved in cell motility. There are three types of cell shape, which are microvilli, lamellipodia, and filopodia.

  • Microvilli are projections on surface that increase surface area.
  • Lamellipodia are membrane ruffles that help sense the environment and direct movement.
  • Filopodia are like microvilli but are less stable. They also sense the environment. They can turn into lamellipodia.

Microfilaments are formed when individual actin monomers polymerize, in a process fueled by ATP hydrolysis, to form chains of filamentous actin. Microfilaments are dynamic structures, growing and shrinking in a controlled manner. Some microfilaments play a structural role in the cell to maintain cell shape. These structural microfilaments have protein caps at both ends to prevent changes in microfilament length. Other microfilaments have functions that require dynamic changes in length. The microfilaments also mediate cytoplasmic streaming, a mixing of the cytoplasm that aids diffusion.

Intermediate filaments edit

Intermediate filaments are polymers of keratin. They help with cell shape also because it bears tension in the cell. It is primarily involved in organelle anchorage. The intermediate filaments also consist of various fibrous proteins that have a diameter of about 10 nm. Intermediate filaments often form a meshwork under the cell membrane and, in cells that lack a cell wall, help impart and maintain cell shape. Intermediate filaments are fairly stable and are not thought to undergo acute changes in length the way microfilaments.

Functions of Cytoskeleton edit

Cytoskeletons are often synthesized based on the cell's needs; however, some protein fibers are permanent. The changeable nature of the cytoskeleton thus contributes to its five important functions.

Cell Shape edit


The mechanical strength of the cell is due to protein scaffolding in the cytoskeleton. In some cells, protein scaffolding also determines the shape of the cell. Microvilli are supported by cytoskeletal fibers such as microfilaments. Microvilli also increase the surface area of the cell for the absorption of materials.

Internal Organization edit

The cytoskeletal fibers of the cell help stabilize positions of organelles. However, the interior arrangement of the cell varies based on the cell's needs. The organelles are dynamic and change minute to minute.

Intracellular Transport edit

The cytoskeleton has the ability to move materials not only in the cell but also within the cytoplasm, thus aiding in the movement of organelles as well. This function is important especially in the nervous system where materials are often transported over long intracellular distances.(3908283293)

Assembly Of Cells Into Tissues edit

Cells are connected to one another through the linking of the protein fibers of the cytoskeleton as well as the protein fibers in the extracellular space. In the process, materials outside the cell are stabilized as well. The assembly of cells not only contributes to the mechanical strength of the tissue but also allows information to transfer between cells from one to another.

Movement edit

Motor Protein

The cytoskeleton of the cell allows the cell to move. For example, the cytoskeleton of white blood cells allows them to squeeze out of blood vessels. Growing nerve cells are also able to send out extensions that allow them to elongate. The microtubule cytoskeleton of cilia and flagella on the cell membrane allow them to move. In addition, motor proteins using energy from ATP can aid in the movement of cells and intracellular transport by sliding along cytoskeletal fibers. There are three types of motor proteins in the cytoskeleton that include myosins, kinesins and dyneins.Kinesins oftentimes brings vesicles from inside the cell out to the periphery. If the kinesins are mutated, then there is likely to be a neural disorder. KIF1β is one disorder resulting from a mutated kinesin. It causes lack of strengths in arms and legs. Dynein, on the opposite hand, brings the vesicles from the periphery back to the inside of cell. Motor proteins convert energy into movement and those found in the cytoskeleton use stored ATP. Myosins allow for muscle contractions. Kinesins allow for the movement along microtubules as dyneins help with the whiplike motion of the microtubule bundles of cilia and flagella. Many motor proteins consist of two heads (to bind to the cytoskeleton fiber), a neck and a tail region at the end to bind to organelles.

Quality control of cytoskeletal proteins edit

The evolution of cytoskeletal proteins edit

The evolution of cytoskeletal proteins required a novel biogenesis machinery. The cytoskeleton in eukaryotes enhances intracellular trafficking and cell division. These functions were once believed to be the distinguish elements of eukaryotes from prokaryotes because bacterial cytoskeleton also composed of proteins that similar to actin and tubulin. The actin-like and tubulin-like proteins in bacteria form filamentous structures which imply in the division of genetic material and maintenance of cell shape.[2] However, actins and tubulins in eukaryotes are distinct from similar prokaryotic proteins in the way that they maintain innovative properties which is critical for eukaryogenesis (the origin of the eukaryotic condition/the evolution of the eukaryotic condition).[3]

Both eukaryotes and bacteria have a cytoskeleton. The bacterial proteins homologous to actin are MreB and ParM, and the bacterial proteins homologous to tubulin are FtsZ. However, actin and tublin differ from MreB, ParM, and FtsZ in the sense that they have properties important for eukaryogenesis.

Actins and tubulins in eukaryotes formed microfilaments and microtubules which unite with their complementary molecular motors (myocin, kinesin, dynein) to be used for phagocytosis. Phagocytosis enables endosymbiosis and also cilia's development which supports mobility and sensory.[4] The inception of actins and tubulins in eukaryotes is an important factor in facilitating their efficient folding and assembly [5][6] which is called cytoskeletal protein biogenesis machinery (CPBM). The CPBM includes molecular chaperones which assist folding of chaperonin containing tailless complex polypeptide-1 (CCT), prefoldin (PFD) - phosducin-like proteins that regulate CCT functions, and five other cofactors. [7][8][9][10][11] In addition, post-translational modifications and proteasomal degradation are required in regulating the function of actins and tubulins which is also unique to eukaryotes.
On the other hand, in prokaryotes, the cytoskeletal protein biogenesis machinery is absent.

Autoregulation of cytoskeletal protein synthesis edit

Actin and tubulin concentrations are strickly controlled due to their critical effects on cytoskeletal dynamics. In animal cells, tubulin synthesis is regulated by an autoregulatory feedback mechanism that can sense the concentration of tubulin heterodimer in order to regulate the stability of ɑ-tubulin, and β-tubulin mRNAs. [12][13] Similar to animals, the synthesis of tubulin in metazoans is also autoregulated due to its critical influences. Researches have shown that overexpression of β-tubulin in Saccharomyces cerevisiae leads to abnormal microtuble function and slow growth.[14]

Actin overexpression causes by an incompletely characterized feedback mechanism that is sensitive to the concentration of actin monomers is preventable by the presence of the 3' untranslated region of actin mRNA.[15]

Biogenesis of cytoskeletal proteins edit

The cytoskeleton protein biogenesis machinery is found in eukaryotes but not in prokaryotes. It includes chaperonin containing CCT (complex polypeptide-1) and PFD (prefoldin), phosductin-like proteins, and five cofactors. The chaperonin molecules help actin and tubulin proteins to fold. Challenges to the protein biogenesis machinery include: (1) the high concentrations of actin and tubulin in cells, (2) the tendency of actin and tubulin to self-associate, (3) the inability of actin and tubulin to fold without the help of other molecules, (4) the fact that actin and tubulin compete with one another for the same folding space.

Biogenesis of cytoskeletal proteins faces a lot of difficulty which mainly due to the abundant of actin and tubulin concentrations, self-associate tendency, and the inability in folding independently. In addition, the competing for access to limited folding space is also a challenge to biogenesis of cytoskeletal proteins.[16] Fortunately, the action of specific chaperonin cofactors can account for the regulation of chaperonin-mediated cytoskeletal protein folding.

Eukaryotic cytosolic chaperonin edit

Eukaryotic cytosolic chaperonin is a unique ability to assist the folding of actin and tubulin. Molecular chaperones can interact with the newly synthesized polypeptide chains to become stable during the folding process form a linear monomer amino acids chain into a more complex and functional protein [17]. There are many types of chaperones that direct the folding of new proteins, refolding of stress-denatured proteins, unfolding of proteins, and transporting proteins [18][19].

Chaperonin, an important family of molecular chaperones, has a barrel-like structure with two multimeric stacked rings of 60kDa (Dalton's atomic mass unit). Chaperonins undergo ATP-dependent conformational changes during its folding cycle: facilitate substrate binding, encapsulation and release[20]. In eukaryotes, the cytosolic chaperonin is the tailless complex polypeptide 1 ring complex (CCT) which is required for viability in yeast and worms.

CCT is crucial for the biogenesis of actin and tubulin. CCT is composed of eight related subunits - ɑ,β,ɣ,ẟ,ɛ,ʝ,ŋ,θ - which show up twice in each oligomer. CCT is closely related to archaeal chaperonin thermosome but quite different from bacterial chaperonin GroEL [21]. CCT is known to have a more specific binding profile than bacterial GroEL [22][23]. As a chaperonin, CCT undergoes ATP-dependent conformational changes during its folding cycle. Due on the copiousness of actin and tubulin, they occupy significant proportion of CCT complexes. Beside actin and tubulin, there are other CCT substrates also have key roles in progression of the cell cycle [24] Researches show that CCT function in vivo is regulated by several dedicated cofactors, including PFD and phosducin-like proteins.

PFD, a jellyfish-shaped molecular chaperone required for stabilization of new cytoskeletal proteins, is a CCT co-chaperone for the biogenesis of actin and tubulin [25][26].

Phosducin-like proteins, regulators of the folding of actin and tubulin in association with CCT, are thioredoxin domain-containing proteins with homology to phosducin, a regulator of retinal G-protein signaling [27].

Tubulin folding cofactors edit

Scientists believed that actin is released from CCT in a native, assembly-competent state, but cyclase associated protein might interact with and stabilize near-native or unstable forms of actin in close association with the chaperonin [28]. On the other hand, functional tubulin is an obligate ɑ-β heterodimer, and evolved in a folding pathway linked to dimer assembly[29].

Roles of each cofactor:
1. Tubulin cofactor A (TBCA): collects unassembled beta-tubulin
2. Tubulin cofactor D (TBCD): assists beta-tubulin down the assembly pathway
3. Tubulin cofactor B (TBCB): binds to alpha-tubulin after chaperonin release
4. Tubulin cofactor E (TBCE): receives alpha-tubulin from TBCB and processes it further
5. Tubulin cofactor C (TBCC): promotes GTP hydrolysis in beta-tubulin if in the presence of a stable supercomplex (formed by the joining of beta-tubulin-TBCD and alpha-tubulin-TBCE complexes)

a. also facilitates the release of native alpha-beta-tubulin heterodimer

Diseases linked to the cofactors:
It is believed that if TBCB is not degraded properly, a neurological disease called giant axonal neuropathy may result. This disease is related to decreased density of the microtubule of cells. Mutations of TBCE are associated with hypoparathyroidism (a developmental disorder), mental retardation, and facial dysmorphism (HRD).

PFD edit

Prefoldin (PFD) is a CCT co-chaperone required for stabilizing nascent cytoskeletal proteins. It consists of two alpha-type and four beta-type subunits which collectively make its structure resemble the shape of a jellyfish. The six subunits form a cavity shaped like a rectangle that attaches to newly formed actin and tubulin as the chains leave the ribosome. PFD then delivers the actin and tubulin to CCT, probably via a docking and substrate-release mechanism (supported by electron microscopy analysis of PFD-actin complexes). It is also possible that PFD improves the efficiency of actin and tubulin protein folding by navigating partially folded molecules back towards the CCT for more folding. This idea is supported by the fact that yeast cells without PFD were observed to fold actin and tubulin more slowly than wild-type cells. Furthermore, Pfd1 knockout mice showed signs of dysfunction of cytoskeletal proteins, neuronal loss, neuromuscular defects, and defective development of lymphocytes. These Pfd1 knockout mice were only viable for five weeks.

Phosducin-like proteins edit

Phosducin-like proteins regulate the folding of actin and tubulin. Three such proteins have been termed PhLP1, PhLP2, and PhLP3 as they are CCT-binding proteins. PhLP1 assists in assembling heterotrimeric G-proteins by CCT; this process is regulated by PhLP1 phosphorylation. PhLP2 and PhLP3 are involved in the biogenesis of cytoskeletal proteins. It is believed that there is a specificity of PhLP2 for actin biogenesis and of PhLP3 for tubulin biogenesis. In other words, disruption of PhLP2 function caused severe actin cytoskeletal defects whereas disruption of PhLP3 function alters normal tubulin biogenesis.

When studied in vitro, it is observed that an excess of PhLP2 and PhLP3 prevents actin and tubulin from being folded via the CCT-mediated folding pathway. It is believed that this is due to the reduced activity of CCT ATPase. However, when the study in done on yeast cells, it appears that PhLP2 stimulates actin folding by purified yeast CCT. The researchers of this study concluded that amino acids of mammalian PhPL2 that were not present in yeast PhLP2 are responsible for preventing actin and tubulin from being folded. This also supports the idea that higher eukaryotes evolved with more regulation of cytoskeletal proteins.

Protein biogenesis/quality control pathways edit

1. Translation

a. Following translation, nascent actin and tubulin undergo folding via:
i. PFD folding pathway.
1. Assisted by PFD and substrate delivery.
ii. PFD-independent pathway.

2. Folding

a. Both PFD and PFD-independent folding pathways lead to formation of CCT (cytosolic chaperonin).
b. CCT-mediated folding follows, leading to:
i. Formation of near-native alpha- and beta-tubulin.
ii. Formation of near-native actin.

3. Assembly, Disassembly, and Polymerization

a. Near-native alpha- and beta-tubulin assembles into a TBCE-TBCD complex.
i. Complex forms folded tubulin heterodimer.
ii. Folded tubulin heterodimer polymerizes into a microtubule.
b. Near-native actin is folded into a folded actin monomer.
i. Folded actin monomer polymerizes into a microfilament.

4. Degradation

a. Free tubulin and free actin not used in polymerization undergo ubiquitylation into proteasome.
i. Proteasome is then degraded.

Post-translational modification (PMT) of cytoskeletal proteins edit

For actin, post-translational modification is known to affect only folding. Tubulin, on the other hand, is affected by PTM in such a way that allows native proteins to turn on and off activity in a reversible and regulatory manner. Tubulin can be modified in a number of ways such as acetylation, detyrosination, and glutamylation. These tubulin modifications occur on microtubules, and it has been hypothesized (though not well-tested) that the free tubulin heterodimer is the substrate responsible for reversing the modifications. Much study, however, has been dedicated to Tubulin PTMs in general. Tubulin acetylation, for example, has been shown in recent studies to be linked to a human neurodegenerative disease called amyotrophic lateral sclerosis (ALS).

Degradation of cytoskeletal proteins edit

Part of the quality control process includes the degradation of proteins. Damaged proteins and misfolded proteins that cannot be refolded with the help of chaperones are removed in this process. Unfortunately, the pathway in which the degradation occurs is not as well studied as the biogenesis process.Invalid parameter in <ref> tag

Turn-over rates and steady-state concentrations of all cellular proteins is regulated in the following ways:

1. protein degradation through the ubiquitin-proteasome system (UPS)

2. lysosomal degradation 3. another proteolytic mechanism

“Proteostasis” is the regulatory process in which damaged or incorrectly folded proteins that cannot be repaired by charperones are removed by proteolysis. Not much work has been put into the study of actin and tubulin degradation, but tubulin has been known to rapidly degrade in the presence of microtubule-destabilizing drugs such as colcemid. Such drugs make tubulin more soluble.

Parkin edit

Parkin is a ubiquitin-protein ligase important to tubulin degradation. Parkin is mutated in patients with autosomal recessive juvenile Parkinson disease (PD). Its normal function is to interact with HSP70-interacting protein (CHIP) to make stress-denatured proteins undergo ubiquitylation. Parkin is also believed to stimulate ubiquitylation and proteasomal degradation of alpha-tubulin and beta-tubulin. Cells that over-express mutant alpha-synuclein, a toxic inclusion-forming protein in Parkinson disease, reveal increased concentrations of alpha-tubulin and insoluble parkin. These two traits are also observed in patients affected by Lewy body disease.

Cofactor E-like edit

E-like (COEL), a tubulin folding cofactor, is a protein that destabilizes tubulin. Cells without human COEL contain excess amount of stable microtubules while microtubule disassembly and the degradation of α-tubulin is observed in cells with excess COELs. The degradation of tubulin caused by the presence of COEL is countered by a negative regulator of microtubule called stathmin that isolates tubulins. Overall, COEL is important for three reasons: 1. it can remove misfolded tubulin 2. regulate the concentration of tubulin 3. control tubulin isotype.[30]

Actin Degradation edit

In metazoan cells, the concentration of tubulin is reduced when CCT or PFD does not function properly. The concentration of actin, however, is not affected significantly. This suggests that the quality control for actin differs from that of tubulin. Relative to the control tubulins, there seems to be less of a need to remove misfolded β-actins. Still, there are incidences when actin degradation is necessary. In the case of ischemic oxidative damage, α-actin specific to the heart is degraded by proteasome. It is also found that α-actins are degraded by lysosomes when muscle contraction in cardiomyocytes is inhibited by the use of drugs. It has been recently observed that TRIM32, an ubiquitin that ubiquitylates α-actin in vitro, when unusually expressed in the human embryonic kidney cells, reduces the concentration of cytoplasmic β-actins. TRIM32 mutations have been found in Bardet-Biedl syndrome and muscular dystrophy, although the actual role TRIM32 plays in these diseases remains unknown.[31]

References edit

Reece, Jane (2011). Biology. Pearson. ISBN 978-0-321-55823-7. {{cite book}}: Text "coauthors+ Lisa A. Urry, Michael L. Cain, Steven A. Wasserman, Peter V. Minorsky, Robert B. Jackson" ignored (help)

Silverthorn, Dee Unglaub. "Compartmentation: Cells and Tissues." Human Physiology. Boston, MA: Pearson Custom Pub., 2007. Print.

  1. Lundin , Victor , Michel Leroux, and Peter Stirling. "Elsevier: Article Locator." | Search through over 10 million science, health, medical journal full text articles and books.., n.d. Web. 17 Nov. 2012. <>.
  2. J. Pogliano The bacterial cytoskeleton Curr. Opin. Cell Biol., 20 (2008), pp. 19–27
  3. M.R. Leroux, F.U. Hartl Protein folding: versatility of the cytosolic chaperonin TRiC/CCT Curr. Biol., 10 (2000), pp. R260–264
  4. T. Cavalier-Smith The phagotrophic origin of eukaryotes and phylogenetic classification of Protozoa Int. J. Syst. Evol. Microbiol., 52 (2002), pp. 297–354
  5. S. Bertrand et al. Folding, stability and polymerization properties of FtsZ chimeras with inserted tubulin loops involved in the interaction with the cytosolic chaperonin CCT and in microtubule formation J. Mol. Biol., 346 (2005), pp. 319–330
  6. S. Bertrand et al. Folding, stability and polymerization properties of FtsZ chimeras with inserted tubulin loops involved in the interaction with the cytosolic chaperonin CCT and in microtubule formation J. Mol. Biol., 346 (2005), pp. 319–330
  7. S. Lacefield, F. Solomon A novel step in beta-tubulin folding is important for heterodimer formation in Saccharomyces cerevisiae
  8. P.C. Stirling et al. PhLP3 modulates CCT-mediated actin and tubulin folding via ternary complexes with substrates
  9. P.C. Stirling et al. Functional interaction between phosducin-like protein 2 and cytosolic chaperonin is essential for cytoskeletal protein function and cell cycle progression
  10. E.A. Mccormack et al. Yeast phosducin-like protein 2 acts as a stimulatory co-factor for the folding of actin by the chaperonin CCT via a ternary complex
  11. M. Lopez-Fanarraga et al. Review: postchaperonin tubulin folding cofactors and their role in microtubule dynamics J. Struct. Biol., 135 (2001), pp. 219–229
  12. D.W. Cleveland et al. Unpolymerized tubulin modulates the level of tubulin mRNAs Cell, 25 (1981), pp. 537–546
  13. M.E. Sellin et al. Global regulation of the interphase microtubule system by abundantly expressed Op18/stathmin Mol. Biol. Cell, 19 (2008), pp. 2897–2906
  14. D. Burke et al. Dominant effects of tubulin overexpression in Saccharomyces cerevisiae Mol. Cell. Biol., 9 (1989), pp. 1049–1059
  15. A. Lyubimova et al. Autoregulation of actin synthesis requires the 3′-UTR of actin mRNA and protects cells from actin overproduction J. Cell Biochem., 76 (1999), pp. 1–12
  16. M.R. Leroux, F.U. Hartl Protein folding: versatility of the cytosolic chaperonin TRiC/CCT Curr. Biol., 10 (2000), pp. R260–264
  17. F.U. Hartl, M. Hayer-Hartl Converging concepts of protein folding in vitro and in vivo Nat. Struct. Mol. Biol., 16 (2009), pp. 574–581
  18. F.U. Hartl, M. Hayer-Hartl Converging concepts of protein folding in vitro and in vivo Nat. Struct. Mol. Biol., 16 (2009), pp. 574–581
  19. E.T. Powers et al. Biological and chemical approaches to diseases of proteostasis deficiency Annu. Rev. Biochem., 78 (2009), pp. 959–991
  20. C.R. Booth et al. Mechanism of lid closure in the eukaryotic chaperonin TRiC/CCT Nat. Struct. Mol. Biol., 15 (2008), pp. 746–753
  21. C. Spiess et al. Mechanism of the eukaryotic chaperonin: protein folding in the chamber of secrets Trends Cell Biol., 14 (2004), pp. 598–604
  22. C. Dekker et al. The interaction network of the chaperonin CCT EMBO J., 27 (2008), pp. 1827–1839
  23. A.Y. Yam et al. Defining the TRiC/CCT interactome links chaperonin function to stabilization of newly made proteins with complex topologies Nat. Struct. Mol. Biol., 15 (2008), pp. 1255–1262
  24. C. Spiess et al. Mechanism of the eukaryotic chaperonin: protein folding in the chamber of secrets Trends Cell Biol., 14 (2004), pp. 598–604
  25. S. Geissler et al. A novel protein complex promoting formation of functional alpha- and gamma-tubulin EMBO J., 17 (1998), pp. 952–966
  26. J. Martín-Benito et al. Structure of eukaryotic prefoldin and of its complexes with unfolded actin and the cytosolic chaperonin CCT EMBO J., 21 (2002), pp. 6377–6386
  27. M. Blaauw et al. Phosducin-like proteins in Dictyostelium discoideum: implications for the phosducin family of proteins EMBO J., 22 (2003), pp. 5047–5057
  28. E.A. McCormack et al. Mutational screen identifies critical amino acid residues of beta-actin mediating interaction between its folding intermediates and eukaryotic cytosolic chaperonin CCT J. Struct. Biol., 135 (2001), pp. 185–197
  29. M. Lopez-Fanarraga et al. Review: postchaperonin tubulin folding cofactors and their role in microtubule dynamics J. Struct. Biol., 135 (2001), pp. 219–229
  30. Lundin, Victor, Loroux, Michel and Stirling, Peter. “Quality Control of Cytoskeletal Proteins and Human Disease” January 2010: 288-295.Retrieved on 20 November 2012.
  31. Lundin, Victor, Loroux, Michel and Stirling, Peter. “Quality Control of Cytoskeletal Proteins and Human Disease” January 2010: 288-295.Retrieved on 20 November 2012.

Berg, Jeremy "Biochemistry", Chapter 35 Molecular Motor. pp1018-1020. Seventh edition. Freeman and Company, 2010.

Slonczewski, Joan L. Microbiology "An Evolving Science." Second Edition. All the different membranous organelles perform different tasks in the cell (animal cells and plant cells). However, they also communicate with each other though ions and lipids. In addition, some of the organelles have membrane that are connected. The others are related by transfer of membrane segments. As we know, structures determine functions in a lot of ways in nature. We can categorize eukaryotic organelles into four categories according to their general functions.

Manufacturer edit

In this first category, nucleus, ribosomes, rough ER, smooth ER and golgi apparatus are included. They can perform this task because of they have metabolically active membranes. Some of them can also perform transportation of the products. Below is some examples of the manufacturing task these organelles do.

  • Nucleus--DNA and RNA synthesis
  • Ribosome--Polypeptide(protein) synthesis
  • Rough ER--Membrane protein synthesis, secretory proteins synthesis and hydrolytic enzymes synthesis; formation of transport vesicles
  • Smooth ER--Lipid synthesis; detoxification and carbohydrate metabolism in liver cells
  • Golgi apparatus--Modificaiton, temporary storage and transportation of macromolecules such as proteins; formation of lysosomes and transport vesicles

Waste disposer edit

In this category, lysosomes, peroxisomes and vacuoles are included. They are all consist of single membrane sac that can be used to collect materials and/or break down unwanted/harmful materials.

  • Lysosomes--Digestion of bacteria, damaged organelle
  • Peroxisomes--breakdown of H2O2 by-product
  • Vacuoles--Digestion of waste products; collect excess water from the cell

Energy provider edit

In this category, chloroplasts and mitochondria are included. They both have metabolically active membranes and intermembrane compartments that can convert energy units to provide to the cells.

  • Chloroplast--Conversion of light energy to chemical energy in plant cells
  • Mitochondria--Conversion of chemical energy of food to chemical energy of ATP

Support, Movement and Communication between cells edit

In this last category, cytoskeleton(such as cilia, flagella and centrioles in animal cells), cell walls(in plants, fungi and some protists), extracellular matrix(in animal cells) and cell junctions are included. They are all responsible for the keeping cells in shape.

  • Cytoskeleton--Maintenance of cell shape; movement of organelles within cells; cell movement; mechanical transmission of signals from exterior of cell to interior
  • Cell walls--Maintenance of cell shape and skeletal support; surface protection; binding of cells in tissue
  • Extracellular matrix--Binding of cells in tissues; surface protection; regulation of cellular activities
  • Cell junctions--Communication between cells; binding of cells in tissues

Overview edit

An overview of plant cell.

Plants are eukaryotes, multicellular organisms that have membrane-bound organelles. Unlike prokaryotic cells, eukaryotic cells have a membrane-bound nucleus. A plant cell is different from other eukaryotic cells in that it has a rigid cell wall, a central vacuole, plasmodesmata, and plastids. Plant cells take part in photosynthesis to convert sunlight, water, and carbon dioxide into glucose, oxygen, and water. Plants are producers that provide food for themselves (making them autotrophs) and other organisms.

Parts edit

These are some of the parts common to plant cells:

Cell Wall- smooth layer that provides DNA and protection from osmotic swelling.

Cell (Plasma) Membrane- it is composed of a phospholipid lipid bilayer (including polar hydrophilic heads facing outside and hydrophobic tails facing each other inside) that makes it semipermeable and thus capable of selectively allowing certain ions and molecules in/out of the cell.

Cytoplasm- it consists of the jelly-like fluid in and around the organelles.

Cytoskeleton- is made up of microtubules, intermediate filaments, and microfilaments. It provides shape the shape of the cell and helps in transporting materials in and out of the cell.

Golgi Apparatus (body/complex)- it is the site where membrane-bound vesicles are packed with proteins and carbohydrates. These vesicles will usually leave the cell through secretion.

Vacuole- stores metabolites and degrades and recycles macromolecules.

Mitochondria- is responsible for cellular respiration by converting the energy stored in glucose into ATP.

Ribosome- contain RNA and proteins for protein synthesis. One type is embedded in Rough ER and another type puts proteins directly into the cytosol.

Rough Endoplasmic Reticulum (roughER)- covered with ribosomes, it stores, separates, and transports materials through the cell. It also produces proteins in cisternae, which then go to the Golgi apparatus or insert into the cell membrane.

Smooth Endoplasmic Reticulum (smooth ER)- it has no ribosomes embedded in its surface. Lipids and proteins are produced and digested here. Smooth ER buds off from rough ER to move newly-synthesized proteins and lipids. The proteins and lipids are transported to the Golgi apparatus (where they are made ready for export) and membranes.

Peroxisome- is involved in metabolizing certain fatty acids and producing and degrading hydrogen peroxide.

Nuclear Membrane (envelope)- the an extension of the endoplasmic reticulum that wraps around the nucleus. Its many gaps allow traffic in/out of the nucleus.

Nucleus - it contains DNA in the form of chromosomes or chromatin and controls protein synthesis.

Nucleolus - it is the site of ribosomal RNA synthesis.

Centrosome- consisting of a dense center and radiating tubules, it organizes the microtubules into a mitotic spindle during cell division.

Chloroplast- conducts photosynthesis and produces carbohydrates, oxygen, and internally ATP and NADPH from captured light energy.

Starch Granule- temporarily stores produced carbohydrates from photosynthesis. Depending on the organism, it can be inside or outside of the chloroplast (if present).

Exclusive to Plant Cells edit

Cell Wall edit

The cell wall is a tough, usually flexible but fairly rigid layer that surrounds the plant cells. It is located just outside the cell membrane and it provides the cells with structural support and protection. A major function of the cell wall is to act as a pressure vessel, preventing over-expansion when water enters the plant cells. The strongest component of the cell wall is a carbohydrate called cellulose, a polymer of glucose.

The cell wall gives rigidity and strength to the plant cells which offers protection against mechanical stress. It also permits the plants to build and hold its shape. It limits the entry of large molecules that may be toxic to the cell. It also creates a stable osmotic environment by helping to retain water, which helps prevent osmotic lysis.

While the cell wall is rigid, it is still flexible and so it bends rather than holding a fixed shape due to its tensile strength. The rigidity of primary plant tissues is due to turgor pressure and not from rigid cell walls. This is evident in plants that wilt since the stems and leaves begin to droop and in seaweed that bends in water currents. This proves that the cell wall is indeed flexible. The rigidity of healthy plants is due to a combination of the cell wall construction and turgor pressure. The rigidity of the cell wall is also affected by the inflation of the cell contained. This inflation is a result of the passive uptake of water.

Cell rigidity can be increased by the present of a second cell wall, which is a thicker additional layer of cellulose. This additional layer can be formed containing lignin in xylem cell walls, or containing suberin in cork cell walls. These compounds are rigid and waterproof, making the secondary cell wall very stiff. Secondary cell walls are present in both wood and bark cells of trees.

The primary cell wall of most plant cells is semi-permeable so that small molecules and proteins are allowed passage into and out of the cell. Key nutrients, such as water and carbon dioxide, are distributed throughout the plant from cell wall to cell wall via apoplastic flow.

The major carbohydrates that make up the primary cell wall are cellulose, hemicellulose and pectin. The secondary cell wall contains a wide range of additional compounds that modify their mechanical properties and permeability. Plant cell walls also contain numerous enzymes, such as hydrolases, esterases, peroxidases, and transglycosylases, that cut, trim and cross-link wall polymers. The relative composition of carbohydrates, secondary compounds and protein varies between plants and between the cell type and age.

There are up to three strata, or layers, that can be found in plant cell walls:

The middle lamella, which is a layer rich in pectins. This is the outermost layer that forms the interface between adjacent plant cells and keeps them together.

The primary cell wall which is generally a thin, flexible layer that is formed when the cell is growing.

The secondary cell wall which is a thick layer that is formed inside the primary cell wall after the cell is fully grown. It is only found in some cell types.

A vacuole inside a plant cell.

Vacuole edit

The vacuole is essentially an enclosed compartment that is filled with water containing inorganic and organic molecules including various enzymes in solution. Vacuoles are formed by the fusion of multiple membrane vesicles and are effectively just larger forms of these vesicles. This organelle does not have a basic shape or size since its structure is determined by the needs of the cell. The functions of the vacuole in the plant cell include isolating materials that may be harmful to the cell, containing waste products, maintaining internal hydrostatic pressure within the cell, maintaining an acidic internal pH, containing small molecules, exporting unwanted substances from the cell, and allowing plants to support structures such as leaves and flowers. Vacuoles also play an important role in maintaining a balance between biogenesis and degradation of many substances and cell structures in the organism. Vacuoles aid in the destruction of invading bacteria or of misfolded proteins that are building up within the cell. They have the function of storing food and assist in the digestive and waste management process for the cell.

Most mature plant cells have a single large central vacuole that takes up approximately 30% of the cell's volume. It is surrounded by a membrane called the tonoplast, which is the cytoplasmic membrane separating the vacuolar contents from the cell's cytoplasm. It is involved in regulating the movements of ions around the cell, and isolating substances that may be harmful to the cell.

Other than storage, the main function of the central vacuole is to maintain turgor pressure against the cell wall. The proteins that are found in the tonoplast control the flow of water into and out of the vacuole through active transport, pumping potassium ions into and out of the vacuolar interior. Because of osmosis, water will flow into the vacuole placing pressure on the cell wall. If there is a significant amount of water loss, there is a decline in turgor pressure and the cell will plasmolyse. Turgor pressure exerted by the vacuole is required for cellular elongation as well as for supporting plants in the upright position. Another function of the vacuole is to push all contents of the cell's cytoplasm against the cellular membrane which helps keep the chloroplasts closer to light.

Plasmodesmata edit

Plasmodesmata are microscopic channels that traverse the cell walls of plant cells enabling the transport and communication between the cells. Plasmodesmata enable direct, regulated intercellular transport of substances between the cells. There are two forms of plasmodesmata, primary ones that form during cell division and secondary ones that form between mature cells. They are formed when a portion of the endoplasmic reticulum is trapped across the middle lamella as a new cell wall is laid down between two newly divided plant cells and this eventually becomes the cytoplasmic connection between the two cells. It is here that the cell wall is thickened no further and depressions or thin areas known as pits are formed in the walls. Pits usually pair up between adjacent cells.

Plasmodesmata are constructed of three main layers, the plasma membrane, the cytoplasmic sleeve, and the desmotubule. The plasma membrane part of the plasmodesmata is an extension of the cell membrane and it is similar in structure to the cellular phospholipid bilayers. The cytoplasmic sleeve is a fluid-filled space that is enclosed by the plasma membrane and is an extension of the cytosol. The trafficking of molecules and ions through the plasmodesmata occurs through this passage. Smaller molecules, such as sugars and amino acids, and ions can pass through the plasmodesmata via diffusion without the need for additional chemical energy. Proteins can also pass through the cytoplasmic sleeve but it is not yet known just how they are able to pass through. Finally, the desmotubule is a tube of compressed endoplasmic reticulum that runs between adjacent cells. There are some molecules that are known to pass through this tube but it is not the main route for plasmodesmatal transport.

The plasmodesmata have been shown to transport proteins, short interfering RNA, messenger RNA, and viral genomes from cell to cell. The size of the molecules that can pass through the plasmodesmata is determined by the size exclusion limit. This limit is highly variable and is subject to active modification. There have been several models that have been proposed for the active transport through the plasmodesmata. One suggestion is that such transport is mediated by the interactions with proteins that are localized on the desmotubule, and/or by chaperones partially unfolding proteins which allows them to fit through the narrow passage.

Plastid edit

Plastids are the site of manufacture and storage of important chemical compounds that are used by the cell. They often contain pigments used in photosynthesis and the types of pigments present can change or determine the color of the cell. Plastids are responsible for photosynthesis, storage of products like starch, and the ability to differentiate between these and other forms. All plastids can be traced back to proplastids, which happen to be present in the meristematic regions of the plant. In plants, plastids may differentiate into several forms depending on what function they need to play in the cell. Undifferentiated plastids, the proplastids, can develop into the following types of plastids:

•Chloroplasts: for photosynthesis

•Chromoplasts: for pigment synthesis and storage

•Leucoplasts: for monoterpene synthesis

The inside of a chloroplast.

Chloroplasts are the organelles that conduct photosynthesis. They capture light energy to conserve free energy in the form of ATP and reduce NADP to NADPH. They are observed as flat discs usually 2 to 10 micrometers in diameter and 1 micrometer thick. The chloroplast is contained by an envelope that consists of an inner and outer phospholipid membrane. Between these layers is the intermembrane space. The material within the chloroplast is called the stroma and it contains many molecules of small, circular DNA (though it is often found in branched linear form, such as in corn). Within the stroma are stacks of thylakoids, which are the site of photosynthesis. The thylakoids are arranged in stacks called grana. A thylakoid has a flattened disk shape and has an empty space called the thylakoid space or lumen. The process of photosynthesis takes place on the thylakoid membrane. Embedded in the thylakoid membrane are antenna complexes that consist of the light-absorbing pigments, such as chlorophyll and carotenoids, as well as the proteins that bind the pigments. These complexes increase the surface area for light capture and allows the capture of photons with a wider range of wavelengths. The energy of the incident photons is absorbed by the pigments and funneled to the reaction center of the complex through resonance energy transfer. From there, two chlorophyll molecules are ionized, which produces an excited electron which passes on to the photochemical reaction center.

Chromoplasts are responsible for pigment synthesis and storage. They are found in the colored organs of plants such as fruit and floral petals, to which they give their distinctive colors. This is always associated with a massive increase in the accumulation of carotenoid pigments. Chromoplasts synthesize and store pigments such as orange carotene, yellow xanthophylls and various other red pigments. The most probably main evolutionary role of chromoplasts is to act as an attractant for pollinating animals or for seed dispersal via the eating of colored fruits. They allow for the accumulation of large quantities of water-insoluble compounds in otherwise watery parts of plants. In chloroplasts, some carotenoids are used as accessory pigments in the process of photosynthesis where they act to increase the efficiency of chlorophyll in harvesting light energy. When leaves change color during autumn, it is because of the loss of green chlorophyll unmasking these carotenoids that are already present in the leaves. The term "chromoplast" is used to include any plastid that has pigment, mainly to emphasize the contrast with leucoplasts which are plastids that have no pigments.

Leucoplasts lack pigments and so they are not green. They are located in roots and non-photosynthetic tissues of plants. They can become specialized for bulk storage of starch, lipid or protein and are then known as amyloplasts, elaioplasts, or proteinoplasts, respectively. In many cell types, though, leucoplasts do not have a major storage function and are present to provide a wide range of essential biosynthetic functions, including the synthesis of fatty acids, many amino acids, and tetrapyrrole compounds such as haem. Extensive networkds of stromules interconnecting leucoplasts have been observed in epidermal cells of roots, hypocotyls and petals.

Introduction edit

The heat stress response (HSR) in plants involves a complex network of different pathways which occurs in different cellular compartments. Scientists have been trying to figure out which is the main thermosensor that initiate the transcription of heat stress response gene in response to the heat stress.Up till now, scientists predicted that there are at least four major thermosensors in plants that activate a similar set of HSR gene which helps improving thermotolerance in plants, include a plasma membrane channel, an unfolded protein sensor in endoplasmatic recticulum, an unfolded protein sensor in cytosol, and a histone sensor in the nucleus. [1] However, the relationship between these four thermosensors remains unknown.

The Significances of Heat Stress in Plants edit

1. Heat plays tremendous roles, especially those of adverse, in plant development, growth, reproduction and fertilization. Plant tissues involved in reproduction are especially vulnerable to the exposure of heat.

2. Plants are sessile organisms that cannot escape heat. Their metabolism is unique as they adapt their metabolism to counterbalance this disadvantage.

3. Cells and organelles in plants are designed in a way to combat and prevent damages caused by heat.

4. Studies for heat stress in plants help agriculture. Necessary measures for heat damage may be prepared from these studies. Under heat stress, programmed cell death can be activated in plants and this can lead to the shredding of leaves, flowers not blooming, little to no production of fruits, or simply the death of plants. In addition, the reproductive tissues in plants are especially sensitive to heat. A difference of a few degrees in temperature can lead to no crops for the season. This can have an enormous and negative impact on the economy.[1] It is estimated that the 1980 and 1988 US heat waves resulted in overall damages of approximately 55 and 71 billion dollars[2]

The Effects of Heat Stress in Plants edit

Heat stress has certain effects on different components in plant cells such as the stability of membranes, proteins, RNA and skeleton structure in cells.It also can change the way chemical reactions are carried out such as enzymatic reactions in the metabolic system. Due to the changes in these components, metabolic process of plant cells becomes imbalance. The disrupted state of metabolites can result in unwanted by-products such as reactive oxygen species (ROS).[1]

In response to the changes of surrounding temperature, plants re-program their transcriptome, protome, metabolome and lipidome. This action basically induces changes in the composition of certain transcripts, metabolites and lipids which help the plants reset a new metabolic balance so that the cells can survive and function as normal even at very high temperature.[1] Moreover, when the temperature comes back to normal, plant cells can reverse the reprogram process and get back to the original metabolic balance that fit in with the current temperature. In addition, plants can also program cell death in response to the heat stress such as resulting in leaves shedding.

Types of Heat Response in Plants edit

Several heat treatments have been applied on plants to study the heat response in plants. There are three major set of heat treatments as mentioned in the article "How do plants feel the heat" by Ron Mittler* : no priming, stepwise priming, and gradual priming.[1]

In the "no priming" treatment, the plants are introduced to a series of severe heat stress. For example, a plant that normally grows at 21oC is placed in an environment at 42-45oC for 0.5 to 1 hour. Plant survival is then measured 5-7 days after the treatment to determine the effect of heat stress.[1] Due to this series of severe heat applied, the plant dies very quickly (exponential decay). The plants ability to survive after such treatment is referred to as basal thermotolerance, the ability of plants to sense and adapt to extreme heat without any priming.

"Stepwise priming" subjects the plants to an environment of moderate heat stress. This is set up by first letting the plants grow at 21oC, and then subjecting them to a temperature of 36-38oC for a short period time of 1.5 hours. They are then recovered to the normal temperature of 21oC for 2 hours after to which they are subjected to a severe temperature of 45oC. Their survivability under such condition (after priming) is known as acquired thermotolerance.

"Gradual priming" is the process which the temperature was increased gradually and steadily until it reaches the temperature equal to extreme heat of 45oC. This is supposed to imitate the conditions that is seen in nature. Plants under these conditions also exhibit acquired thermotolorence. For Arabidopsis thaliana, gradual priming increase the survival rate by more than 10%. It is preferred over both no priming heat stress and stepwise priming temperature changes. [3]

The last type of heat treatment is "warming", which is not considered as one of the heat stress treatments. During warming treatment, plants are originally at a temperature lower than standard growing temperature of 12oC, instead of 21oC. They are then exposed to a temperature that is slightly higher than their ordinary growth temperature so instead of subjecting them to 21oC, they are grown at 27oC. This slight increase in temperature change from the norm is, however, not considered heat stress since plants actually do not express HSR markers under warming. In general, warming allows longer term adaptation and reprogramming of the development, which includes early flowering or shedding leaves.

The important message to take home from this experimental setup is that different heating treatments elicit different transcriptome responses. This suggests that there may be separate heat sensors and signaling pathways activating specific responses to rise in temperature.

Sensing heat in plants edit

When an A. thaliana experiences an increase in external temperature, its large surface-to-volume ratio makes sure that almost all macromolecules such as protein complexes, membranes and nucleic acid polymers in the plant cells to acknowledge the heat immediately. As it is true for every matter in the universe, the macromolecules' kinetic energy is raised by the introduction of heat into the system. This, consequently, results in reversible physical changes of the macromolecules. There are certain macromolecules that not only just "perceive" heat but also differentially trigger a unique signaling path that can specifically upregulate hundreds of HSR genes[4]

There are 2 basic routes plants use to mediate heat stress 1. Direct effect in a specific sensor: The specific macromolecule that behaves as a sensor molecule could be directly affected by heat. It is observed when temperature-induced changes in its quaternary and tertiary structures occur. 2. Indirect effect in a specific sensor: In this route, the specific macromolecule sensor is indirectly influenced by heat due to the effects of heat on other components of the cell. For example, temperature-induced changes in membrane fluidity could affect a membrane protein which is embedded in. It is indirect because the membrane protein is actually affected by heat but is only subject to change when the fluidity of plasma membrane varies.

Heat sensing at Plasma Membrane edit

The fluidity of the plasma membrane can increase when there is an increase in temperature. Scientists have found out that the increased fluidity of the plasma membrane will activate calcium channels, which will lead to an influx of Calcium ions into the cell. This is the primary heat sensing event in the moss Physcomitrella patens.[1] This is the mechanism of detecting heat. However, the actual heat stress sensor in plants cells in still unknown. The A. thalianagenome encodes over 40 calcium channels and many of them are known to be located in the plasma membrane. These presences of calcium channels strongly suggest that calcium ions are the ones, which are responsible for sensing heat.

Beside activating the Calcium channels, membrane fluidity change might also trigger lipid signaling due to the change of temperature.

How does a heat stress-induced inward calcium flux can control regulate multiple signaling pathways in plants?

In A. thaliana, the following 4 pathways are known to happen:

Mechanism of protein kinases

1. The calmodulin AtCaM3 signals heat stress and it itself is involved in the activation of different transcription factors such as WRKY39[5] and HSFs [6]

2. Chain reactions may be triggered by heat signaling. It is known that an inward flux of calcium activates unique calcium-dependent protein kinases or the ROS-producing enzyme NADPH oxidase. A protein kinases is a kinase enzyme that modifies other proteins by chemically adding phosphate groups to them.

3. AtCaM3 activated calcium/calmodulin-binding protein kinase. This protein kinase phosphorylates HSFA1a.

4. An HSP90/FKBP-dependent kinanse can also mediate HSF phosphorylation causued from calcium binding to calmodulin [7]

Lipid Signaling edit

On top of ion channel activation in the plasma membrane, membrane fluidity changes caused by increase heat can also trigger lipid signaling. During heat stress, phospholipase D (PLD) and phosphatidylinositol-4-phosphate 5-kinase (PIPK) are activated. Lipid signaling molecules such as phosphatidic acid, D-myo-inositol-1,4,5-trisphosphate (IP3), and phosphatidylinositol-4-phosphate (PIP2) start to accumulate from heat stress and tigger the calcium channels open, resulting in calcium influx. On a side note, it should be noted that a reduction in phospholipase C9 activity is correlated with lower concentration of IP3, sHSP downregulation, and lower thermotolerance.

The mechanistic relationship between lipid signaling response to heat stress and plasma membrane channels (whether it is direct or indirect) is still unclear. The chronological order of events in heat stress sensing and signaling response also remains as a question to be answered. It is likely that the signaling pathways function downstream to what's responsible for sensing heat stress in plasma membrane.

Change in Histone occupancy edit

Histones are proteins that help package DNA. As seen in wild-type plants, the increase in temperature causes a significant decrease in concentration of certain histones such as H2A.Z which contains the nucleosomes that might trigger the changes in transcriptome so that HSR genes are initiated. Scientists also found that the decrease in H2A.Z histone concentration might also cause the change in the expression of a specific transcription factor or other regulatory proteins which can initiate the response of transcriptome. After screening for mutants impaired in heat sensing, it was discovered that the ARP6 gene is possibly responsible for mediation of responses to temperature changes. ARP6 codes for a SWR1 subunit that is required in order for H2A.Z to be inserted into nucleosomes, instead of H2A. It was shown that warming in wild-type plants result in significant decrease in H2A.Z occupancy in nucleosomes where the transcription for warming-induced genes starts. Since less histones are in the way, this allows for more transcriptions of these warming-induced genes. Less H2A.Z occupancy in certain HSP promoters can also affect transcription factor and other regulatory proteins' expression and DNA binding ability, ultimately inducing transcriptome response. It is, however, unsure whether the occupancy of the histones is the cause for heat sensing related to acquired thermotolerance. [5].

Unfolded Protein Response edit

Heat stress may activate the unfolded protein response (UPR) in both ER and Cytosol which can initiate heat stress response(HSR). The UPR is the weakening of protein stability. In the ER pathway, unfolded proteins can enter the nuclei and therefore affect the transcription of certain genes. This will lead to an accumulation of ER chaperone transcripts which will alter the metabolism of the cell in order to adapt to the heat.[1] Beside heat stress, UPR can also be activated by specific chemicals that cause UPR or changes in certain abiotic aspects.[8] Because few unfolded proteins and HSR chaperones are likely to exist under mild heat conditions, the UPR is considered to be not as sensitive as the PM heat response.[1]

ROS Signaling edit

Different metabolic pathways are likely to depend on enzymes with different responsiveness to unnecessary heat. From this reason. it has been proposed that heat stress might uncouple some metabolic pathways and cause the accumulation of unwanted by-products. An example of such by-product is reactive oxygen species (ROS). Studies have shown that the accumulation of ROS is mostly likely due to the change in fluidity of the plasma membrane.[9] This event is most likely a positive feedback loop since the accumulation of ROS will also open up more calcium channels in the plasma membrane and therefore lead to more calcium influx into the cell.[1] Therefore, it seems that the plasma membrane heat sensing is highly interlinked with ROS signaling. A large accumulation of ROS may lead to program cell death.[1]

Conclusion edit

Although scientists have proposed possible ways in which plants can sense heat, there is still much we don’t understand about the specific mechanisms and the order of events. How the signals from the speciated sensors integrate with each other within the network of signal transduction is also still of an important question that is yet to be answered. There could be additional pathways not yet discovered but crucial to heat stress response.

References edit


  1. a b c d e f g h i j k Finka, A., Goloubinoff, P. and Mittler, R.. “How Do Plants Feel the Heat?” Trends in Biochemical Sciences. March 2011: 118-125.
  2. "Genetic engineering for modern agriculture: challenges and perspectives" by R. Mittler, E. Blumwald
  3. Invalid <ref> tag; no text was provided for refs named Larkindale
  4. "How do plants feel the heat?" by Ron Mittler, Andrija Finka, Pierre Goloubinoff
  5. "Functional characterization of Arabidopsis thaliana WRKY39 in heat stress" from Mol. Cell, 29 (2010), pp 475-483) by S. Li
  6. "The role of class A1 heat shock factors (HSFA1s) in response to heat and other stress in Arabidpsis" from Plant Cell Environment, 34 (2011), pp738-751 by H.C. Liu
  7. "Arabidopsis ROF1(FKBP62) modulates thermotolerance by interacting with HSP90.1 and affecting the accumulation of HsfA2-regulated sHSPs" from Plant Journal 59 (2009), pp 387-399 by D. Meiri, A Breiman
  8. Moreno, A.A. and Orellana A. (2011) The Physiological role of the unfolded protein response in plants. Biol. Res. 44, 75-80.
  9. Königshofer, H. et al. (2008) Early events in signaling high-temperature stress in tobacco BY2 Cells involve alterations in membrane fluidity and enhanced hydrogen peroxide production. Plant Cell Environ. 31, 1771-1780.
  10. Kumar, S.V. and Wigge, P.A. (2010) H2A.Z-containing nucleosomes mediate the thermosensory response in Arabidopsis. Cell 140, 136–147

Overview edit

Cellulosomes are a complex of multi-cellulolytic enzymes that break down cellulose and hemicelluloses found in the cell walls of plants. Cellulose and hemicelluloses are two organic polysaccharide compounds that are also two of the most abundant polymers on Earth. They are major sources of carbon and energy. Cellulases and hemicellulases participate in protein:carbohydrate [carbohydrate-binding modules (CBMs)] or protein:protein (dockerins) interactions that make it so hard to break down. Chemically, cellulose is relatively simple. However its crystalline structure makes it resistant to biological degradation. Deconstruction and digestion of cellulose and hemicelluloses will allow for carbon turnover and numerous biotechnological uses, including biofuels as an alternative fuel source. However, very few microorganisms have the capacity to digest cellulose and hemicelluloses, which are why cellulosomes are of great interest.

Discovery edit

The first cellulosome was discovered in the early 1980s by Bayer and Lamed in their studies of the cellulolytic system of Closteridium thermocellum, an anaerobic bacterium. They described the cellulosome as a “discrete, cellulose-binding, multi-enzyme complex that mediates the degradation of cellulosic substrates.” They initially believed that the cellulosome only degraded cellulose, but it was later recognized that the cellulosome also degrade a large number of hemicellulases and even pectinases. Also following Bayer and Lamed’s studies were research in identifying the molecular mechanisms of how the enzyme complex assembles and how the cellulosome is present on the surface of the host bacterium. More recently, a range of anaerobic bacteria and fungi were shown to also produce cellulosome systems.

Function edit

Scientists have argued that cellulosomes are more efficient in digesting cellulose and hemicellulases than corresponding enzyme systems produced by aerobic bacteria and fungi. It is possible that anaerobic environments imposed selective pressures that led to the evolution of cellulosomes. One hypothesis proposes that the integration of plant cell wall-degrading enzymes onto a macromolecular complex leads to a more efficient enzyme complex whose cellulosomal catalytic units interactions work together synergistically and further enhanced by enzyme substrate targeting through scaffoldin-borne CBM. Another hypothesis suggest that the function of the cellulosome on the surface of the bacterium, at least in C. thermocellum, enhances the capacity of the host bacterium to utilize mono- and oligosaccharide released from the cell wall.

General Structure edit

The cellulosome was found to be composed cellulosomal catalytic components that contained noncatalytic modules called dockerins, which bind to cohesin modules. This receptor/adapter protein domain pair is responsible for cellulosome self-assembly. The recognition between cohesin and dockerin is type and/or species specific. Together cohesin and dockerin are located in a large noncatalytic protein that acts as a scaffoldin.

Dockerins edit

Dockerins consists of approximately 70 amino acids that contain two segments, each of about 22 residues, in the cellulosomal catalytic components. The first 12 residues resemble the calcium-binding loop of EF hand motifs in which aspartate or asparagines are highly conserved. The EF hand is a helix-loop-helix structural domain found in a large family of calcium-binding proteins. Calcium was shown to be necessary for dockerin stability and function. Without calcium, dockerins are unable to interact with cohesions. They are also present in a single copy at the C-terminus of cellulosomal enzymes.

Cohesins and Scaffoldins edit

Cohesins are 150 residue modules of tandem repeats in scaffoldins, which specifically bind to dockerin modules. The number of cohesin modules in scaffoldins varies between one and eleven, but is usually more than four. Scaffoldins can be defined as cohesin containing proteins, which play a role in cellulosome assembly. The scaffoldin subunits of the cellulosome function to organize and position other protein subunits into the complex and can also serve as an attachment device for harnessing the cellulosome onto the cell surface and/or for targeting its substrate because scaffoldins usually contain a noncatalytic CBM that anchors the entire complex onto cellulose.
More recently, researchers found that some cellulosome-producing microbes produce more than one type of scaffoldin. Scaffoldins that bind cellulases and hemicellulases are called primary scaffoldins. Proteins that bind primary scaffoldins are called anchoring scaffoldins.

How Cellulosomes Bind to Plant Cell Walls edit

Cell-surface attachment of cellulosomes is required for degradation of plant cell wall polysaccharides. CBMs are carbohydrate-binding modules that are separated into three types: Type A (interacts with crystalline polysaccharides), Type B (bind to internal regions of single glycan chains), and Type C (recognizes small saccharides). It was first thought that bacterial cellulosomes are bound to the cellulose by the “cellulose-binding factor,” but it was later realized that the attachment of the cellulosome to the cell wall is mediated by a family 3 CBM (CBM3) located in the scaffoldins. CBM3s are type A modules that bind tightly to the cellulose surface.

Bacterial Cellulosomes edit

There are two major types of bacterial cellulosomes: those present in multiple types of scaffoldins and those that contain a single scaffoldin. Cellulosomes that assemble via a single primary scaffoldin are the simplest and contain six to nine catalytic components, depending on the number of cohesions in the primary scaffoldin. Bacterial cellulosomes are bound tightly to the cellulose and referred to as the “cellulose-binding factor.”
Bacteria expressing cell-surface cellulosomes contain a single primary scaffoldin and multiple anchoring scaffoldins. The majority of the anchoring scaffoldins contain SLH modules or SLH domains, which mediate the attachment of the structural proteins to the bacterial cell wall and may bind to the secondary cell wall polysaccharides.

Fungal Cellulosomes edit

Anaerobic fungi can hydrolyze many different polysaccharides, including cellulose and hemicelluloses. It is believed that anaerobic fungi are the initial invader of lignocelluloses and play a role in fiber digestion along with bacteria and other microorganisms. Anaerobic fungal plants have cellulosomes just like anaerobic bacteria. However, fungal cellulosomes are less characterized.
A number of dockerin sequences have been identified in different fungi strains. Fungal dockerins consist of a three-stranded beta-sheet and a short helix held together by two disulfides. Researchers also found that the amino acid sequences of fungal dockerins are unrelated to their bacterial counterparts. Genome sequencing of fungi will allow scientists to see which dockerin sequences present in proteins are associated with plant wall degradation. Another difference between fungal and bacterial cellulosomes is that fungal cellulosomes generally contain two copies of dockerin modules, each consisting of approximately 40 residues joined together by shorter linker sequences.
Researchers have also hypothesized that fungal cellulosome may not involve cohesin modules. However, more work and research is needed in characterizing fungal cellulosomes.

Applications edit

Studying cellulosomes and how they work would provide a large number of biotechnical benefits to society. There are many industrial applications, such as paper pulp production, that requires breakdown of plant cell walls in its process. Cellulosomes would be a great benefactor.
One of the biggest challenges facing the world today is the lack of alternative and renewable energy sources to the conventional fossil fuels. A potential, viable alternative can be found in the converting lignocellulosic biomass to fermentable sugars to produce renewable fuels, such as ethanol. Yet, the biggest rate limiting problems in converting lignocelluloses into biofuels, is the hydrolysis of structural polysaccharides, which require the development of a more efficient enzyme system. Extensive enzyme group is needed to overcome the recalcitrant and the plant cell wall. Cellulosomes could be the future solution.


References edit

  1. 1. Fontes, Carlos and Harry J. Gilbert. “Cellulosomes: Highly Efficient Nanomachines Designed to Deconstruct Plant Cell Wall Complex Carbohydrates.” Annual Review of Biochemistry. 2011.

Overview edit

An animal cell is a type of cell that dominates most of the tissue cells in animals. Animal cells are different from plant cells because they don't have cell walls and chloroplasts, which are relevant to plant cells. Without the cell wall, animal cells can be in any sort of shape or size as they are instead surrounded by a plasma membrane.

One thing why animal cells are exclusive because they have centrioles and plant cells do not have centrioles. Centrioles are important for DNA segregation when the cell undergoes the process of mitosis, a process of dividing a cell. Centrioles are important in the structure of the spindles, which helps to pull the chromosomes apart.

Both animal and plant cells have vacuoles, however, in animal cells, vacuoles are very tiny or absent while the vacuole in plant cells are quite large. There are different kinds of animal cells such as- muscle cells, nerve cells, and many more.

Parts edit

Cell Membrane: The cell membrane is a fluid mosaic structure which is composed of a phospholipid bilayer and other important macromolecules such as proteins. The cell membrane separates the cell from the environment and allows the movement of materials in and out of the cell.

Animal Cell

Cytoplasm: the liquid within the cell where the different organelles are found. It is here where many functions occur. Including cell division and glycolysis.

Golgi Apparatus: The organelle in which proteins are modified, sorted, and sent to various parts of the cell. Modifications on the protein include but are not limited to, glycosylation.

Mitochondria: does the cellular respiration of the cell by converting glucose into ATP (cellular energy).

Ribosome: The mRNA from the nucleus are used by Ribosomes in a process called translation. Translation is when the Ribosome joins amino acids together according to the sequence of the mRNA. The more ribosomes in a cell, the proteins it synthesizes. They are located in two areas, on the ER or in the cytosol.

Rough Endoplasmic Reticulum:: is used to store and transport material through the cell. Proteins are produced here in the ribosomes bound to the rough ER.

Smooth Endoplasmic Reticulum: Functions in the synthesis of lipids, detoxification of drugs and poisons, storage of calcium ions, and metabolism of carbohydrates. In contrast to the Rough Endoplasmic Reticulum, the smooth ER is not studded with proteins.

Peroxisome: A specialized metabolic compartment bounded by a single membrane. Additionally, it possesses enzymes that transfer hydrogen atoms from substrates to oxygen, producing hydrogen peroxide as a by-product. Then, hydrogen peroxide is converted to water by another enzyme.

Nucleus: The nucleus is usually the largest organelle in a cell. It consists of different parts such as the nuclear envelope, chromosomes, and the nucleolus. The nuclear envelope surrounds the nucleus while segregating the chromatin from the cytoplasm and consists of two membranes each made of a lipid bilayer. The membranes have pores that regulate what goes in and out of the nucleus. Inside the nucleus is the nucleolus which holds the genetic material DNA. Using this DNA, transcription is carried out making mRNA.

Vacuole: the "storage space" that stores water, salt, and other important substances. There are also food vacuoles that are cellular organelles in which food is broken down by hydrolytic enzymes. These food vacuoles are the simplest digestive compartments. The process of intracellular digestion occurs inside vacuoles, which is the process of hydrolysis of food. This process begins after a cell engulfs food materials through phagocytosis (solid food) or pinocytosis (liquid food).

Lysosome: considered the "digestion compartment" of the cell. Lysosomes break down cellular wastes such as fats, proteins, or carbohydrates. The rid of the cellular materials that are no longer useful in the cell.

Cytoskeleton: is a structure made out of protein to give the cell its shape and structure. It also helps cellular motion with the use of flagella, cilia, or lamelllipodia.

Centrioles: are used through cell division. They organize the mitotic spindle during the end of cytokinesis. The centrioles are located within the centrosome and come in pairs. Each pair of centrioles are compiled of nine sets of triplet microtubules assembled into a ring. Prior to animal cell division, the centrioles replicate. Although centrosomes with centrioles may assist the organization of microtubule construction in animal cells, they are not crucial for this particular function in all eukaryotes; e.g. the fungi and the majority of plant cells lack centrosomes with centrioles, but still contain well-assembled microtubules. [1]

References edit

  1. Biology 9th edition, Campbell

Overview edit

Schematic Diagram of the MSCs in a Eukaryotic Cell

Two organelles in a cell come in contact via membrane contact sites (MCS). Membrane contact sites govern the signaling and crossing of ions and lipids from one cellular organelle to the next. Even though research on MCS have been going on for over 50 years now, there is still so much more to uncover regarding their function, structure and regulation. To this date, there are multiple studies done on three main contact sites: the nucleus-vacuole junction, mitochondria-ER, and plasma membrane-ER contact sites. Having membrane- bound organelles is a special characteristic for eukaryotic cells, as it allows for specialized functions in different compartments of the cell. Two organelles exchange information through interorganellar or MCS when they are in close proximity. To visualize their communication, a technique such as electron microscopy was employed. This technique first arose in the 1950s when the close apposition of endoplasmic reticulum and mitochondria was first seen. Ever since then, similar electron microscopy techniques were used to visualize contact sites in cells between the endoplasmic reticulum and: mitochondria, vacuoles, Golgi, chloroplasts and plasma membrane. Since most studies were conducted without molecular markers, they usually refer to proximity of around 30 nanometers between two organelles’ membranes. Unfortunately, there is still no excellent 3D model visualization of MCS. However, in the recent years, there is an increase in the studies of the proteins that reside in the membrane sites and molecular determinants needed to create MCS.

The Nucleus-Vacuole Junction (NVJ) edit

The membrane contact site between the nucleus and the vacuole is known as the nucleus-vacuole junction (NVJ). The primary study is of the yeast cell Saccharomyces cerevisiae, in which the vacuolar membrane, inner nuclear membrane, and the ER membrane is bridged through NVJ. Nucleus-vacuole junctions are the best studied MCS to this date. NVJ is made of vacuolar protein Vac8 and the ER-localized protein Nvj1. If one of these two proteins is missing from deletion of genes, the MCS will not be made and there would not be contact between two organelles.
 : There have been two cellular functions suggested for the NVJ. The first one is piecemeal microautophagy of the nucleus. This is a process that marks a part of the yeast nucleus for degradation in the vacuole lumen. Piecemeal microautophagy of the nucleus is stimulated during nitrogen or carbon starvation and requires autophagic parts shared between the electrochemical gradient across the vacuolar membrane, the selective cytoplasm to vacuole, and unselective microautophagy. The second suggested function of NVJ is lipid biosynthesis. The two proteins that participate in lipid biosynthesis, Osh1 and Tsc13, reside in the NVJ. Osh1 comes from the yeast oxysterol-binding protein homology family whose role includes lipid sensors and lipid transfer. However, there is no evidence that shows NVJ regulates lipid biosynthesis because localization of osh1 in NVJ is exclusive to stationary phase. In addition, OSH1 is not needed for forming NVJs or for the piecemeal microautophagy of the nucleus in starved cells. The other protein resides in NVJ (Tsc13) is responsible for catalyzing the terminal step in biosynthesis of very-long-chain fatty acids(VLCFAs). These fatty acids can affect the structure and fluidity of membranes. Although we have pretty good understanding of these two enzymes, we haven't figured out why they reside in NVJ or if there are other proteins that are in the NVJ.
 : Similar contact sites between ER and the endosome/lysosome (for eukaryote cells) have been observed. Instead of osh1, late endosome-localized oxysterol-binding protein-related protein 1L(ORP1L) works as a cholesterol sensor and it sense level of cholesterol by changing its conformation. Through the change of conformation, ORP1L tells ER how much cholesterol there is in the cell.
 : Although NVJ is the most studied membrane contact site, we still don't know how much it can do for a cell.

The Mitochondria-ER contact site edit

The second most-studied MCS is the mitochondria-ER site. This site is primarily visualized via electron microscopy. The section of ER that co-purifies with the mitochondria, known as mitochondria-associated membrane fraction (MAM), is researched extensively on. The two main functions of this site is lipid biosynthesis and Ca2+ homeostasis. Early research showed that the MAM fraction encompasses enzymes that participate in phospholipid biosynthesis. In addition, the mitochondria and ER must work together to make certain lipids. For example, let’s take a closer look in the biosynthetic pathway of phosphatidylcholine. The substrate for this pathway is made in the ER, which then enters the intermembrane space of the mitochondria to produce phosphatidylethanolamine (PE), and then PE goes back to the ER to synthesize phosphatidylcholine. This process shows that the ER and mitochondria collaborate to produce certain lipids. On the other hand, ER-mitochondria MSCs is responsible for the transmission of Ca2+ . Homeostasis of mitochondrial Ca2+ is important because Ca2+ regulated the ATP production rate. Furthermore, an overload of Ca2+ can trigger cell death.

The PM-ER contact site edit

There are many contact sites of ER-PM in different cell types. The function of ER-plasma membrane contact sites is similar to that of the mitochondria-ER junctions, since Ca2+ homeostasis and lipid synthesis and trafficking take place there. As mentioned above, Ca2+ regulation is crucial as it is also responsible for protein synthesis, folding, and signaling. In excitable cells, the global calcium signal is generated by the coupling of PM depolarization and ER calcium release. In non-excitable cells, calcium influx is controlled by detecting luminal ER Ca2+ levels. Research has showed that the PM-ER contact site is involved in non-vesicular lipid trafficking. We know that lipids are insoluble in water. So to transfer lipid from its synthesis sites to its desired work place, one must shield the lipid. In fact, couple families of lipid transfer protein (LTPs) that can perform this task have been found on the contact sites. One of them is oxysterol-binding protein (OSBP) related proteins(ORPS). The oxysterol-binding protein related protein is of a big LTP family which is conserved from yeast to man. It binds to sterols and scientist believe it helps to transfer non-vesicular sterol between the ER and PM. Even though PM-ER contact sites is crucial for Ca2+ and lipid signaling, we still cannot identify the tether proteins that mediate such contact. One strong possible protein for PM-ER contact sites is a class of highly conserved protein called junctophilins. It can stabilize the junctions by anchoring ER/SR to the PM, giving a structual basis for physiological coupling between PM and ER/SR Ca2+ channels. Further investigation is necessary to map the molecular machinery for the regulation and generation of PM-ER contact sites.

Conclusion of the Membrane Contact Sites (MCS) edit

Functions of MCS was established first as a site of trafficking of lipids and ions. However, as more and more studies done on MCS, we know that MCSs are also responsible for signaling, effective transmission of metabolic cues within cell, organellar inheritance control mechanism. We are not clear on how MCS perform these tasks yet but better understanding of MCS would be achieved once we figure out its molecular components.

References edit

1. Elbaz, Yael, and Maya Schuldiner. Staying in touch: the molecular era of organelle contact sites. Israel: Elsevier Ltd, 2011. Print.

An interesting look at a paper by Carolyn A Larabell and Keith A Nugent edit

Much knowledge pertaining to cell architecture and its redevelopment during normal and disease filled processes has been derived from imaging. For centuries, the use of light microscopy techniques has reached its peak and has yielded a tremendous amount of information concerning cell and molecular dynamics. There exist two main types of microscopies, of which include fluorescence microscopy and transmission light microscopy. Though these two techniques have significantly advanced our understanding of cellular processes, they tend to provide limited details. Due to the low penetrating capabilities of electrons, it is not possible to examine the eukaryotic cell as a whole. Thus, most cells are sectioned into 60-500 nm slices. This tedious process requires initial fixation, dehydration, and plastic embedment as well as application of heavy metal stains to generate contrast. As can be seen, extensive work is required to obtain three-dimensional structural information of a whole cell, which is why most studies tend to be limited to small sections of cells.

All this leads to the emergence of X-ray imaging technologies, a crucial new tool for cellular imaging. X-rays are versatile, for they can penetrate thick cells and tissues, eliminating the need to section the specimen further. There are in fact three main types of X-ray imaging techniques, of which include soft X-ray microscopy, soft X-ray tomography, and coherent diffraction imaging. X-ray microscopy has the ability to penetrate thick cells deeply and initiate immediate structural development and outlook. Soft x-ray tomography can generate quantitative three-dimensional images of cells in the near-native state (at a better than 50 nm isotropic resolution). The most unique technique, which goes by the name of coherent diffraction imaging (the name speaks for itself), offers the possibility of high resolution x-ray imaging of cell using computation and high speed computers rather than an X-ray optic to phase the image. Let us take a closer look at the specifics of each X-ray imaging technique. Soft X-ray microscopy involves the use of particular zone plate lenses to allow the release of X-rays towards the cell of interest. These microscopes are operated using photons with energies in the ‘water window’, which is the region of the spectrum that lies between the K shell absorption edges of carbon and oxygen. However, lens-based imaging can also with energies outside the water window, where images can be generated using phase contrast techniques. To date, the most gripping images have been obtained using bright field, absorption contrast at a water window wavelength operation. To further specify the operations of each plate optic, it can be said that a condenser zone plate optic focuses X-rays on the cell itself and an objective zone late optic focuses the transmitted light onto the detector.

Soft x-ray tomography is in a sense similar to light and electron microscopes in that it can also generate two-dimensional representations of a three dimensional specimen. However, with the ability to collect projection images of the cell at angles around a rotation, it is possible to mathematically compute a three-dimensional reconstruction of the specimen of interest. It has been noted that all biological materials are damaged when exposed to intense light. This intensity can either come from ultra violet illumination in a fluorescence microscope or photons from an X-ray microscope. However, by cryosplicing, optical beams can be focused at a much lower temperature, thus avoiding the possibility of radiation cell damage altogether. As a matter of fact, when cells are imaged at liquid nitrogen temperatures, more than a thousand soft X-ray projection images can be retrieved without any sort of radiation damage. As stated before, it is crucial that specialized cryogenic tomography stages occur during this process of X-ray imaging. A key fact illustrating soft X-ray tomography’s versatility is that is can be applied to virtually any imaging investigation in cell biology, from imaging simple bacteria, to yeast and even algae. The first recorded evidence of X-ray tomographic reconstruction was algae. The distinctive ability to obtain high resolution images of intact eukaryotic cells is something that cannot be repeated by any sort of light or electron microscopy. In addition, x-ray tomography can be used to examine even the most complex cells, such as white blood cells and malaria-infested red blood cells.

The last, but certainly not least, technique of discussion is the method of coherent diffractive imaging. It is simply a method that offers the possibility of removing the need for a lens, thus avoiding the limitations imposed by the state-of-the-art in fabrication technology. As basic arrangement of a CDI experiment can be seen as a coherent beam which is used to illuminate the sample and its far-field diffraction pattern is measured. The beam will typically interact quite weakly with the sample, and the undiffracted component of the beam is held to be dominant and will damage the detector unless a beam-stop is initiated. The measurement of the beam at very low diffraction angles closely follows this previous process. Instead of a lens, a zone plate is used instead in coherent diffraction imaging, as it is used to create a focus and the sample is placed in the beam diverging from it. Sure enough, soft X-ray microscopy and soft X-ray tomography seem to be more in use at this time, but coherent diffraction imaging has great promise in the future. The procedural techniques must be sharpened further for it to play a bigger role in three-dimensional imaging. After all, CDI offers access to the quantitative amplitude and phase information that are not easily available through other forms of X-ray imaging. One thing is for sure: X-ray imaging techniques will seek development in the years to come.

Hard X-ray Fluorescence Tomography edit

Hard X-ray Fluorescence Tomography was a method originally created to detect the presence of very small amounts of transition metals in biological tissue, like copper, zinc, magnesium, etc. Metal ions like zinc and magnesium, for example, act as cofactors in many catalytic enzymatic reactions, and are hence very important for the cell. Using hard X-rays, a large number of transition metals can be detected and mapped all at once, since transition elements automatically fluoresce when hit by hard X-rays. Although this method is very powerful, there are and have been a number of technical problems with the usage of this method.

Hard X-ray tomography is a technique that uses a very large number of two-dimensional pictures of slices of a tissue in order to assemble a three-dimensional picture of the given sample of tissue. There are three main broad categories of tomography – full-field, projection tomography and confocal tomography. Full-field projection tomography is the process of utilizing the entire field of the specimen to carry out the tomography. However, this is still a young, developing technology with many problems. Projection tomography on the other hand, is moderately successful, and certain errors have to be accounted for in the final image produced. Confocal tomography is the most interesting of the three – it gives scientists the ability to analyze a very small portion of a specimen, although it is difficult to find the particular target using this method.

There are a few challenges being faced by scientists using this method to analyze biological tissues. The first is that the hard X-rays are very strong, and hence very harful to the tissue being studied. This results in only hard tissues like seeds being able to be studies, since other tissues die off so easily under exposure. Samples currently analyzable are freeze-dried or chemically fixed to endure the radiation. Scientists also face a number of time constraints due to speed limitations in dealing with the technical difficulties of using this technique.

Reference edit

Imaging cellular architecture with X-rays. Nugent and Carolyn A Larabell. Hard X-ray fluorescence tomography —an emerging tool for structural visualization. Martin D de Jonge and Stefan Vogt.

Overview edit

Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. It is a form of luminescence. In most cases, the emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation. However, when the absorbed electromagnetic radiation is intense, it is possible for one electron to absorb two photons; this two-photon absorption can lead to emission of radiation having a shorter wavelength than the absorbed radiation. The emitted radiation may also be of the same wavelength as the absorbed radiation, termed "resonance fluorescence". The most striking examples of fluorescence occurs when the absorbed radiation is in the ultraviolet region of the spectrum, and thus invisible to the human eye, and the emitted light is in the visible region.

Role in cell structure edit

Fluorescent stains reveal structures within the cell dramatically and can allow us to see cellular structure better which can lead to better understanding.[1]

References edit

  1. U.S. Department of Health and Human Services. Inside the Cell. September 2005. <>.

Definition edit

The decrease in the cell's ability to proliferate with the passing of time. Each cell is programmed for a certain number of cell divisions and at the end of that time proliferation halts. The cell enters a quiescent state after which it experiences cell death via the process of apoptosis.

Overview edit

Aging is inevitable as it is an accumulation of damage over time that eventually affects the function and survival of organisms. It is evident that this occurs because the accumulation of damages is contributed by the inability of the biological systems to maintain and protect the somatic tissues over a long duration of time. There are approaches that contribute to aging. One approach is outlined by the degradation of physiological systems output which directly leads to functional decline. Functional decline changes in cell number which affects metabolic record of the cell. This simple pathway serves as a framework for analyzing methods involved in aging. Physiological and Metabolic Processes All organisms’ functions are described as a set of physiological systems that interact with each other and the environment. Their system only communicates with other systems through “inputs” and “outputs”. The diagram on the left shows the network of the physiological system that gives a overview of organism’s function. System aging happens over time when output becomes inappropriate and impairs organisms’ function. The progressive dysfunction with aging of an organism is due to the decline influenced by some other outside forces. Physiological Decline From an evolutionary perspective, natural selection will try to maximize reproductive success in tissues in order to slow down mortality. Inversely, many physiological systems within an organism will wear out at similar rates. Similar rates of functional decline could occur because the output of the dysfunctioning system maybe directly be connected to other systems. Another possible explanation for analogous rates would be the existence of an aging process common to different physiological systems. For example, mutations in a single gene ameliorate many forms of aging-related damages. Furthermore, the evolutionary effects on aging raises the probably of similar aging process in different organisms. Cell Number Cell number change is directly correlated to aging. Change in the outputs of a system is influenced by alterations in their constituent cells. Even changes outside the extracellular matrix of exoskeleton can be ascribed to changes in cell function. Accumulation of extracellular debris such as fatty plaques can cause dysfunction of macrophages. Even age related modification such as glycation-mediated loss of elasticity of blood vessel wall or damage to lens protein may contribute to the effectiveness of cell function. Since modifications occur at same hierarchical level as cell changes, they inherently affect system function in much the same way as cell function. Therefore, the decline in physiological systems during aging is caused by changes in cell count and/or changes in noncellular component of the system. Metabolic Control Analysis (MCA) and Aging MCA has been used to describe the control and regulation of metabolic pathways and networks. Activity of each enzymatic step is varies very slightly and the effect of the overall flux is determined. Simple definition of the control is where the greater the change in flux, the greater the control of that enzyme over the flux. Thus, the control lover flux is a property of pathway that can share control and distribution of control as metabolic conditions are altered. Further research is still required in this field of study but scientists have built a concrete foundation in which they can experimentally build upon.

Major Forms of Cell Death edit

There are three main forms of death that are widely recognized: Apoptotic death, Autophagic death, and Necrotic death. Apoptotic death is also known as the programmed cell death. It occurs when cells are no longer needed and they “commit suicide by activating a intracellular death program”. [4] Autophagic death is also another type of cell death, commonly known as autophagic Programmed Cell Death (PCD). [5] This type of death occurs by the delivery of autophagic vesicles into lysosomes [2]. Necrosis death is a more abrupt form of death that cells undergo. Necrosis is when the plasma membrane of a cell brakes. It is important to take into account that these types of deaths could not be exactly the cause of cell death but could happen before, or after.

Premature Cell Death by Phagoptosis edit

Phagocytosis is important because it is a major homeostatic mechanism in multicellular organisms. For example, it defends against pathogens by removing defective cells. However, excessive or lack of cell death is harmful and can lead to pathology. Recent discoveries have shown that cells that are still considered viable can be marked for phagocytosis prematurely in a process known as phagoptosis. Prior to this discovery, phagocytosis was believed to only eat dead cell or cells that are close to death. However, recent studies show that healthy cells can be marked for phagocytosis if they possess a signal marking it for phagocytosis or lack a signal to prevent it from being eaten by phagocytosis. The phagoptosis of neurons is particularly concerning due to the limited capacity of the brain to replace these neurons. By the same logic, prevention of phagoptosis of cancer cells are also of concern.

Phagocytosis ( process of cell devouring) is started by the release of signals (‘ find me’ signals) which attracts macrophages that are close by to the cell. When the macrophage is close enough to the cell it recognizes signals of ‘eat me’ and ‘don’t eat me’ from its surface. The most common “eat-me” signal is generated when cell surface is exposed to phospholipid PS. PS exposure occurs when aminophospholipid translocase (enzyme that assists proteins to move across a membrane) is inhibited or phospholipid scramblase is stimulated or both. Healthy cells usually have aminophospholipid translocase that uses ATP to keep phospholipid PS in the inner plasma membrane. When this translocase is inhibited, PS flows out to the outer membrane and is exposed to cell surface, thereby creating “eat-me” signals. Moreover, any condition that activates phospholipid scramblase, which changes phospholipid distribution on the plasma membrane, can cause PS exposure. Conditions such calcium elevation, ATP depletion, oxidative stress can stimulate scramblase and inhibit the translocase. However, exposure of PS is not the sole indicator on whether or not a cell is marked by a phagocyte and additional environmental conditions must also be met. In addition of having an "eat-me" signal of exposed PS, the cell can also posses a "don't-eat me" signal which can be expressed by the protein CD47. Cells can expose this CD47 "do-not-eat" region causing the binding of certain residues to the protein, resulting in the phagocyte being unable to bind to the protein. Moreover, not all cells that are PS-exposed are sufficient to start phagocytosis. Some cells have strong “don’t eat me” signal or require other more potent signals to induce phagocytosis. Therefore, phagocytosis depends on the net exposure of both types of signals and the amount of PS or CD47 expressed determines if phagocytosis occurs. PS exposure can be reversible if and only if the induced eat me signal on the cell surface is quickly removed before macrophage finds the cell. [2]

File:PS exposure on cell surface.jpg
PS exposure on cell surface

The most applicable connection is to current cancer research. Unsurprisingly, cancer cells possess an overwhelming amount of "don't-eat me" or CD47 signals. It was recently shown that high levels of CD47 have a strong correlation to the capability of producing tumors in both mice and humans. Antibodies that identify CD47 have shown to induce phagocytosis of these cancer cells and show promise in future cancer treatment.[2]

Phagoptosis of Neurons edit

LDS activates microglia to release MFG-E8 to bind onto exposed PS of the neuron cell marking it for phagoptosis.

Destruction of neurons can be a serious potential health risk if the natural process goes amiss. The cells responsible for the phagocytosis of living neurons are microglia, which are brain cell macrophages. Microglia cells become activated by lipopolysaccharide (LPS) to consume and destroy inflamed neurons via phagocytosis. This activation of the microglia cells, in essence, makes them hunt for inflamed neurons by discharging MFG-E8, a binding protein. When living neurons become inflamed, they bare the phospholipid phosphatidylserine (PS) on the exterior surface of the cell. Once the PS is out in the open, MFG-E8 binds to PS marking it for the microglia to come engulf and destroy the neuron.[3]

Scientists have been looking for a way to inhibit or block this targeting and destruction of healthy neurons. In one experiment they use mice that have been genetically altered to deactivate a specific gene that is used to make MFG-E8 binding proteins. Their results showed that the mice without the MFG-E8 binding protein did not have phagoptosis of regular inflamed neurons. However, by adding pure MFG-E8 binding proteins back into those mice, phagoptosis of the inflamed neurons resumed as usual. They also found that if LPS is inserted in close proximity to the neurons of a mouse that inflammation would occur and microglia targeted the neurons. Additionally to support their findings, when LPS was inserted into the MFG-E8 deficient mice neuron destruction was much less than it was in the normal mice. [3]

In conclusion, inflammation activates microglia cells to destroy healthy neurons but this can be averted by inhibiting the marking of neurons for destruction. This is not to say that microglia are troublesome, they actually help clean up dead and dying neurons to keep inflammation down. In turn, inflammation would make the microglia go on the hunt for living neurons as well as neurons that need to be disposed of. [3]

Cancer edit

It has been found that cancers usually expose a great quantity of “don’t eat me” signals in order to not undergo cell death, this being the reason why cancer is so difficult to fight. The “don’t eat me” signal majorly exposed by cancers is the CD47 which has been found to have a correlation with tumourigenicity and its exposure amount. [2] The “don’t eat me” signal is usually neautralized by our bodies by the CRT(Cell Surface Calreticulin) “ eat me” signal, which is greatly exposed in the human body.

Programmed Necrotic Cell Injury edit

Necrotic cell death has been revealed to be more coordinated than random as was previously understood. Known as ‘programmed’ necrotic cell injury, this form of cell death is regulated by TNF receptors which are involved in apoptosis as well. Necrotic cell injury differs from apoptosis in that it is characterized by extensive swelling, plasma membrane rupture, and distinct biochemical components involved in the pathway-specifically RIP serine/threonine protein kinases. Apoptosis and programmed necrosis are therefore induced in response to similar cellular conditions with minor disparities in the degree of cell damage as cells communicate and coordinate the proper course of action through molecular cross talk involving structural modification at the cell membrane and signalling between cells. [6]

Reference edit

[1]Toward a Control Theory Analysis of AgingAnnual Review of BiochemistryVol. 77: 777-798 (Volume publication date July 2008) First published online as a Review in Advance on March 4, 2008DOI: 10.1146/annurev.biochem.77.070606.101605Murphy, Michael. Partridge, Linda.

[2]Eaten alive! Cell death by primary phagocytosis: 'phagoptosis'. Brown GC, Neher JJ. Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, UK.

[3]Brown, Guy C. and Neher, Jonas J..”Eaten alive! Cell death by primary phagocytosis: ‘phagoptosis’.” Trends in Biochemical Sciences 37.8 (2012): 325-332. Print.

[4]Alberts, Bruce. "Apoptosis Is Mediated by an Intracellular Proteolytic Cascade." Programmed Cell Death (Apoptosis). U.S. National Library of Medicine, 18 Feb. 0000. Web. 29 Oct. 2012. <>.

[5] Nature Publishing Group, n.d. Web. 29 Oct. 2012. <>.

[6]Moquin D, Chan FK. "The molecular regulation of programmed necrotic cell injury." Trends Biochem Sci. 2010 Aug;35(8):434-41. Epub 2010 Mar 26. Review.

Introduction edit

Cell adhesion is fundamental in the formation of tissues due to the attraction between individual cells. These adhesions are due to the internal cytoskeleton, which determines the overall structure of the cell. The cell can be in two states - stable adhesion interactions and dynamic adhesive interactions. The stable adhesion interactions are typically made up of cell adhesion receptors (which usually are the glycoprotein that binds the ECM between cells), ECM (extracellular matrix, which are large proteins that interact with other cellular receptors on the site) proteins, and cytoplasmic plaque proteins.

Formation of Tissue edit

One of the most significant cell adhesions that is required to form a stable tissue is called the Cadherins adhesion molecules, which are transmembrane receptors that depends on Ca2+ to recognize other cells during growth. One of the most studied cadherin molecules is called E-cadherin, which is known to fight epithelial cancer. Cadherin work by combining with catenins and the actin in the cytoskeleton. Catenins are cytoplasmic plaque proteins, which incorporates both a-catenins and b-catenins. a-catenins are responsible for the linkage between the cadherins to the actin in the cytoskeleton. b-catenins are responsible for the interaction between the a-catenin and the cadherin cytoplasmic domain. Based on the observations of b-catenin's interaction with growth factors and transformation, it can be concluded that b-catenin are simply acting as the regulatory component. Cadherins are also responsible for the regions of the cell known as the junctional localization, which are the stronger points of the cell that is capable for adhesion. The EC (extracellular matrix) domains are split into five sub-domains, EC1, EC2, EC3, EC4, and EC5. With the help of x-ray crystallography, the EC1 sub-domain can be used to form a dimer, more specifically called the strand dimer, which are monomers that are parallel to the adhesive binding surfaces and orient outwards.

Desmosomal Junctions edit

Desmosomal junctions are primarily located in epithelia and cardiac muscles. They link with the intermediate filament cytoskeleton to form a network capable of withholding immense weight.

References edit

  • Gumbiner, Barry M (1996). "Cell Adhesion: The Molecular Basis of Tissue Architecture and Morphogenesis". Cell. 84 (3): 345–57. doi:10.1016/S0092-8674(00)81279-9.
Comparison of Eukaryotes vs. Prokaryotes

Eukaryotes Originated from Prokaryotes edit

Similar gene sequences between prokaryotes and eukaryotes suggest that they originated from a universal ancestor and evolved into separate domains billions of years ago. Prokaryote evolved to eukaryote through several stages. An ancestral anaerobic (without air) eukaryote cannot metabolize efficiently due to its inability to oxidized fuel completely. To improve its metabolism, the ancestral eukaryote ingests a bacterial genome that is aerobic (with air). An aerobic metabolism is more efficient because fuel is oxidized to carbon dioxide. Once the bacterium is engulfed by the eukaryote, it uses the cell for replication. This symbiotic system can now carry out aerobic catabolism; thus, transforming anaerobic eukaryote to aerobic eukaryote. There are three major changes that occurred as prokaryotes evolved to eukaryotes. First, the mechanism needed to fold DNA into compact structure containing specific proteins and the ability to divide equally between daughter cell during cell division became more elaborate. Since cell are now larger, system intracellular membranes developed to create a double membrane to surround the DNA. Lastly, early eukaryotes were incapable of carrying out photosynthesis or aerobic metabolism until an aerobic bacterium is introduced to form endosymbiotic and to eventually form plastids. They are similar in their metabolic reactions and in the way they produce energy, as well as in regards to what both prokaryotic and eukaryotic cells are composed of. To name some of their similarities, both have their cells surrounded by plasma membranes, both contain cytoplasm, and they both contain structures of RNA and protein called ribosomes. Though variations are present, their distinct differences result from DNA mutations that have occurred over time.

Eukaryotic Cells Evolved from Simple Precursors

Major changes of the simple cells lead to the development of Eukaryotic Cells

1. Cells were able to acquire more DNA, therefore mechanisms that required to fold DNA strands into complexes with more specific proteins and divide into daughter cells (cell division)

2. Growth of Cells- the cells grow larger in size allowed intramolecular membranes to develop, which led the development of double membrane surrounding DNA -RNA synethsis on DNA template from cytoplasmic process of protein synthesis on ribosomes became possible

Prokaryotes edit

  • Structure:

    • Cell membrane: phospholipid bilayer that encloses the cytoplasm, serves as attachment point for the intracellular cytoskeleton and cell wall.
    • Cell wall: rigid, outside of the plasma membrane. Its function is to determine the shape of the organism and to act as a vessel pressure, preventing over-expansion when water enters the cell.
    • Nucleoid: analog to nucleolus of eukaryotes, nucleoid contains DNA, genetic material of the cell, but it is not enclosed by any membrane.
    • Chromosomes: contains genetic information. Chromosomes make up nucleoid. Prokaryotic cells are haploid.
    • Flagella: tail-like organelles in charge of movements of cells.
    • Pili: shorter and thinner than flagella, used also for motility and adherence.
  • Morphology of prokaryotic cells

Prokaryotic cells have a variety of shapes. These shapes are to describe, classify and identify micro-organism. Some common shapes are:

    • Cocci: spherical shape
    • Bacilli: cylindrical or rod shape
    • Spirilla: a curves rod long enough to form spirals
    • Vibrio: a short curved rod (comma) shaped
    • Spirochete: long helical shape
  • Cell division
    • Prokaryotic cells reproduce through asexual reproduction. They usually are divided by binary fissions (breaking in half, forming two identical daughter cells) or budding (daughter cells grow out of the parent and gradually increase in size)
    • Prokaryotic cells have their genes passed out completely to their daughter cells through mitosis. Genome is stored in chromosome.
  • Energy intake
  • Bacteria and Archaea are the main branches of prokaryote evolution.
    • Generally, Bacteria and Archaea are quite similar in size and shape. They share the characteristics of prokatyotes but are different in many key structural, biochemical, and physiological characteristics.

Characteristics Bacteria Archaea Nuclear envelope Absent Absent

Membrane-enclosed organelles Absent Absent

Peptidoglycan in cell wall Present Absent

Membrane lipid Unbranched hydrocarbon Some branched hydrocarbons

RNA polymerase One kind Many kinds

Initiator amino acid for start of protein synthesis Formyl-methionine Methionine

Response to antibiotics Growth-inhibited Growth not inhibited

Histones associated with DNA Absent Present

Circular chromosome Present Present

Ability to grow at temperature >100C No Some species

Prokaryotes vs. Eukaryotes Cell edit

Eukaryotic Cell Prokaryotic Cell
Nucleus Present Absent (nucleoid)
# of Chromosomes More than one One - but not a true chromosome; Plasmids present
Cell Type May be unicellular or multicellular Unicellular
True Membrane-bound Nucleus Present (Lysosomes, Golgi-complex, Endoplasmic Reticulum, Mitochondria, Chloroplasts) Absent
Telomeres Present (Linear DNA) Circular DNA; does not need telomeres
Genetic Recombination Mitosis, fusion of gametes Partial, un-directional transfer of DNA
Lysosomes/Peroxisomes Present Absent
Microtubules Present Absent (rare)
Edoplasmic Reticulum Present Absent
Mitochondria Present Absent
Cytoskeleton Present Present
DNA associated with proteins Yes No
Ribosomes Larger (80S); 70S in organelles due to symbiosis Smaller (70S)
Vesicles Present Present
Golgi Apparatus Present Absent
Mitosis Yes No; binary fission
Chloroplasts Present in plants Absent; chlorophyll is scattered in the cytoplasm
Cell Size 10-100 µm 1-10 µm
Permeability of Nuclear Membrane Selective not present in cell
Cell Wall Present on Plant and Fungi cells (chitin) Present (peptidoglycan)
Vacuoles Present Present
Flagella Present; for movement Present; for propulsion

Campbell NA, Reece JB. 2008. Biology. 8th ed. San Francisco (CA): Pearson/Benjamin Cummings.

There are many difference between prokaryote and eukaryote cell. In nuclear body of prokaryote, the nuclear body is not bounded by a nuclear membrane while eukaryotic cell have a nuclear that is bounded by a nuclear membrane having pores connecting it with the endoplasmic reticulum. In prokaryote, the cell is covered by cell envelope, a structure varies with type of bacteria, while in eukaryote cell, there is cell membrane to separate the cell from outside environment and regulates movements of materials in and out of the cells. Circular, unorganized DNA molecule is located in nucleoid of prokaryote; on the other hand, linear DNA that is organized by histones is located in nucleus of eukaryote which the protector of nuclear envelope. In prokaryotes, the nuclear body contains a circular chromosome with the lack of histones (unwounded DNA). There is no nucleolus eukaryotic chromosome but a nucleolus, which is present with one or more paired, linear chromosome containing histones. This says that the DNA of eukaryotic cells are organized in nucleus and the DNA of prokaryotes cells are unorganized and floats free in the nucleolus. Eukaryotes sex cell and prokaryotes cell both have flagella, organelle that helps the cell move. Prokaryote's flagella is consist of two protein building block while eukaryote's flagella is complex and consisting of multiple microtubules. Eukaryotes have many organelles in cells such as mitochondria, golgi, lysosomes.... besides ribosomes, there is no organelles in prokaryotes. Prokaryotic cell on average are usually ten times smaller than eukaryotic cell. Cell division in prokaryotic cell and eukaryotic cell is also different. In prokaryotic cell, the cell divided by binary diffusion and prokaryotic cell are haploid. In eukaryotic cell, cell division follows process of mitosis; haploid sex cells in diploid. Cell membrane in prokaryotic cell is a phospholipid bilayer which usually lacking sterols while eukaryotic cell membrane contains sterols. Eukaryotic cell membrane is capable of endocytosis and exocytosis while prokaryote cell is not. Cell wall is present in plant cell, algea, and fungi which belong to eukaryote. Cell wall of eukaryotic cell never composed of peptidoglycan. In prokaryotic cells, a few member of domain bacteria have cell walls which composed of peptydoglycan. Member of domain Archae have cell wall composed of protein or unique molecules resembling but not the same as peptidoglycan. In cytoplasmic structures of eukaryote, the ribosomes are composed of a 60S and a 40S subunit forming an 80S ribosome. Internal membrane-bound organelles such as mitochondria, endoplasmic reticulum, Golgi apparatus, vacuoles, and lysosomes are present. Chloroplasts serve as organelles for photosynthesis. A mitotic spindle involved in mitosis is present during cell division. A cytoskeleton is present. It contains microtubules, actin micofilaments, and intermediate filaments. These collectively play a role in giving shape to cells, allowing for cell movement, movement of organelles within the cell and endocytosis, and cell division. In prokaryote, the ribosomes are composed of a 50S and a 30S subunit forming an 70S ribosome. Internal membrane-bound organelles such as mitochondria, endoplasmic reticulum, Golgi apparatus, vacuoles, and lysosomes are absent. There are no chloroplasts. Photosynthesis usually takes place in infoldings or extensions derived from the cytoplasmic membrane. There is no mitosis and no mitotic spindle in prokaryote but fission and budding only. They may contain only actin-like proteins that, along with the cell wall, contribute to cell shape.

Eukaryotes edit

  • Structure
    • Plasma membrane: A lipid/protein/carbohydrate complex, providing a barrier and containing transport and signaling systems.
    • Mitochondrion: Surrounded by a double membrane with a series of folds called cristae. Functions in energy production through metabolism. Contains its own DNA, and is believed to have originated as a captured bacterium.
    • Cytoskeleton
      • Microfilaments
      • Intermediate filaments
      • Microtubules
    • Nucleus: double membrane surrounding the chromosomes and the nucleolus. Pores allow specific communication with the cytoplasm. The nucleolus is a site for synthesis of RNA making up the ribosome.
      • Nuclear envelope: doubled membrane, enclosing the nucleus.
      • Nucleolus
      • Chromatin: contains genetic information of cells (DNA)
        • Chromosomes: only visible during cell divisions.
    • Endoplasmic Reticulum (ER)
      • Rough ER: A network of interconnected membranes forming channels within the cell. Covered with ribosomes (causing the "rough" appearance) which are in the process of synthesizing proteins for secretion or localization in membranes.
      • Smooth ER: A network of interconnected membranes forming channels within the cell. A site for synthesis and metabolism of lipids. Also contains enzymes for detoxifying chemicals including drugs and pesticides.
    • Golgi apparatus: A series of stacked membranes.
    • Lysosome: A membrane bound organelle that is responsible for degrading proteins and membranes in the cell, and also helps degrade materials ingested by the cell.
    • Ribosome: Protein and RNA complex responsible for protein synthesis
  • Cell division
  • Energy intake

Eukaryotes that live in extreme environments tend to be microbial. Microbial life is present in all extreme environment with enough energy to support life. Eukaryote cells are very adaptable, but prokaryotes are a little bit more so. This is not conclusive due to many environments that have yet to be explored. Anaerobic environments lack oxygen, but eukaryote cells still manage to survive.

Thermophile eukaryote cells are resistant to high temperatures. These cells must adapt by modifying their lipid and cell walls. The heat is dangerous so modifications to resist heat are necessary.

Psychrophiles are cells that have adapted to extremely cold temperatures. An example would be Heteromita globosa, which is a heterotrophic flagellate that lives in Antarctica. Psychrophiles face an extra challenge in that they must make modifications to the lipid and cell walls in order to maintain fluidity. The cold temperatures cause cell walls to become rigid.

Acidophiles are cells that can survive in extremely acidic conditions. Until recently, there were only four organisms, all eukaryotic that could survive in near 0 pH conditions. The way acidophiles can survive in such low pH levels is not known, but it is suggested that it could be due to a strong proton pump or low proton membrane permeability.

Alkalophiles are cells that can survive in high pH levels. Some of these Alkalophiles can also be stable at neutral pH also which leads us to believe that the internal chemistry is highly resistant to environmental influence. Possible compartmentalization of eukaryotic cells could help this.

Barophiles are cells that survive in high pressure conditions.

Xerophiles are cells that survive in dry conditions.

Halophiles are cells that live in high saline conditions. These cells must face the problem of drying out by osmotic pressure. Eukaryotes such as flagellates thrive in such conditions.


  1. extreme conditions, November 20, 2012.

CRISPR Defense System in Prokaryotes edit

Possible mechanism for CRISPR

The CRISPRs, or Clustered Regularly Interspaced Short Palindromic Repeats, functions as a prokaryotic immune system. It protects bacteria and archaea against mobile genetic elements. This defense system can continuously adjust its reach at the genomic level which implies that both gain and loss of information is inheritable. The CRISPR defense system consists of stretches of interspaced repetitive DNA and associated cas genes. The CRISPR system consists of three distinct stages:

  1. Adaptation of the CRISPR via the integration of short sequences of the invaders as spacers;
  2. Expression of CRISPRs and subsequent processing to small guide RNAs;
  3. Interference of target DNA by the crRNA guides.

CRISPR Mechanism edit

Exogenous DNA is processed into small elements by proteins encoded by CRISPR-Cas genes. These small elements are then inserted into the CRISPR locus near the leader sequence. RNAs from the CRISPR loci are constituitively expressed and processed by cas proteins to small RNAs composed of individual exogenously-derived sequence elements with flanking repeat sequence. The RNAs then guide other cas proteins to silence exogenous genetic elements at both the RNA and DNA level.

Stage 1 - CRISPR adaptation
Adaptation of the CRISPR is the recognition of alien DNA by dedicated cas proteins and/or host proteins, as well as the subsequent processing and integration into the chromosomal CRISPR locus. This stage has recently been demonstrated experimentally by Barrangou et al. by triggering virus resistance on the lactic acid bacterium Streptococcus thermophilus. The acquisition of new spacers occurred at the 5' end of the CRISPR as predicted. The fact that new spacers are integrated at the 5' side of the CRISPR suggests that they are a chronological record that reflects previous encounters with mobile genetic elements. Furthermore, spacers in both sense and anti-sense orientation turned out to be functional while only the leader strand is transcribed. This implies that the mechanism does not operate with a classical anti-sense mechanism at the level of mRNA, implying that DNA is the target.

Stage 2 - CRISPR expression
CRISPR expression is the transcription of the poly-spacer precursor crRNA which is followed by binding to a complex of Cas proteins and processing to mono-spacer crRNAs that serve as the guide sequences. It was discovered that pre-crRNA (longer transcripts) potentially covered the entire CRISPR and were step-wise processed to small crRNAs. Furthermore, only the leader strand of the CRISPR is transcribed and processed. Similar results has been obtained by expression studies of CRISPRS from both the bacterium Staphylococcus epidermidis and the archaea Archaeoglobus fulgidus and S. solfataricus.

Stage 3 - CRISPR interference
CRISPR interference involves the binding and/or degradation of the target nucleic acid (DNA). Studies of CRISPRs from S. thermophilus has provided experimental evidence that the CRISPR-Cas system has the ability to bring about specific viral resistance. Furthermore, experiments on the CRISPR-Cas system from S. epidermidis has showed that this bacterium lost its CRISPR-based immunity against a plasmid after a self-splicing intron was inserted into the proto-spacer of the plasmid. The intron is present at DNA level but spliced from mature RNA. This strongly suggests that DNA is the target of the CRISPR interference system.

Significance for evolution and possible applications
By implementing the CRISPR-Cas mechanism, bacteria are able to acquire immunity against certain phages and halt further transmission of targeted phages. Because of this, some researchers have suggested that the CRISPR-Cas system is a Lamarckian inheritance mechanism.
Several proposals to exploit CRISPRs-derived biotechnology include:

  • Artificial immunization against phages by adding matching spacers of industrially important bacteria, such as in the milk and wine industries.
  • Knockdown of endogenous genes by transformation with a plasmid which contains a CRISPR area with a spacer, which inhibits a target gene.
  • Distinguish different bacterial strains by comparison of the spacer regions.

Reference edit

  • Trends in Biochemical Sciences Volume 34, Issue 8, August 2009, Pages 401-407

DNA-Binding Proteins Distinguish Specific Sequences of DNA edit

The method prokaryotes use most often when responding to environmental changes is altering their gene expression. Expression is when a gene is transcribed into RNA and then translated into proteins. The two types of expression are constitutive—where genes are constantly being expressed—and regulated—where specific conditions need to be met inside the cell for a gene to be expressed. This sub-section focuses on how prokaryotes go about regulating the expression of their genes.

Transcription, when DNA is converted into RNA, is the first place for controlling gene activity. Proteins interact with DNA sequences to either promote or prevent the transcription of a gene.

Keep in mind that DNA sequences are not discernible from one another in terms of having features that a regulatory system would be able to register. Therefore, when regulating gene expression, prokaryotes rely on other sequences within their genome, called regulatory sites. These regulatory sites are most often also DNA-binding protein binding sites and are close to the DNA destined for transcription in prokaryotes.

An example of one of these regulatory sites is in E. coli: when sugar lactose is introduced into the environment of the bacterium a gene for encoding the production of an enzyme, β-galactosidase, begins to be expressed. This enzyme’s function is to process lactose so that the cell can extract energy and carbon from it.

lac regulatory site

The sequence of nucleotides of this regulatory site (pictured) displays an almost completely inverted repeat. This shows that the DNA has a nearly twofold axis of symmetry, which in most regulatory sites usually correlates to symmetry in the protein that binds to the site. When studying protein-DNA interactions, symmetry is generally present.

Furthering the investigation into the expression of the lac regulatory site and the protein-DNA interactions that take place there, scientists looked at the structure of the complex formed between the DNA-binding unit that recognizes the lac site and the site itself, which is part of a larger oligonucleotide. They found that the DNA-binding unit specific to the lac regulatory site comes from the protein lac repressor. The function of lac repressor, as the name suggests, is the repression of the lactose-processing gene’s expression. This DNA-binding unit’s twofold axis of symmetry matches the symmetry of the DNA, and the unit binds as a dimer. From each monomer of the protein an α helix is inserted into the DNA’s major groove. Here amino acid side chains interact (via very site-specific Hydrogen bonding) with the exposed base pairs in such a fashion that the lac repressor can only very tightly bind to this specific site in the genome of E. coli.

The helix-turn-helix motif is common to many prokaryotic DNA-binding proteins edit

After discerning the structures of many prokaryotic DNA-binding proteins, a structural pattern that was observed in many proteins was a pair of α helices separated by a tight turn. These are called helix-turn-helix motifs and are made of two distinct helices: the second α helix (called the recognition helix) lies in the major groove and interacts with base pairs while the first α helix is primarily in contact with the DNA backbone.

In Prokaryotes, DNA-Binding Proteins Bind Explicitly to Regulatory Sites in Operons edit

Looking back at the previous example with E. coli and β-galactosidase, we can garner the common principles of how DNA-binding proteins carry out regulation. When E. coli’s environment lacks glucose—their primary source of carbon and energy—the bacteria can switch to lactose as a carbon source via the enzyme β-galactosidase. β-galactosidase hydrolyzes lactose into glucose and galactose which are then metabolized by the cell. The permease facilitates the transport of lactose across the cell membrane of the bacterium and is essential. The transacetylase is on the other hand not required for lactose metabolism but plays a role in detoxifying compounds that the permease may also be transporting. Here we can say that the expression levels of a group of enzymes that together contribute to adapting to a change in a cell’s environment change together.

An E.coli bacterium growing in an environment with a carbon source such as glucose or glycerol will have around 10 or fewer molecules of the enzyme β-galactosidase in it. This number shoots up to the thousands, however, when the bacterium is grown on lactose. The presence of lactose alone will increase the amount of β-galactosidase by a large amount by promoting the synthesis of new enzymes rather than activating a precursor.

When figuring out the mechanism of gene regulation in this particular instance, it was observed that the two other proteins galactoside permease and thiogalactoside transacetylase were synthesized alongside β-galactosidase.

An operon is made up of regulatory components and genes that encode proteins edit

The fact that β-galactosidase, the transacetylase, and the permease were regulated in concert indicated that a common mechanism controlled the expression of the genes encoding all three. A model called the operon model was proposed by Francois Jacob and Jacques Monod to explain this parallel regulation and other observations. The three genetic parts of the operon model are (1) a set of structural genes, (2) an operator site (a regulatory DNA sequence), and (3) a regulator gene that encodes the regulator protein.

In order to inhibit the transcription of structural genes, the regulator gene encodes a repressor protein meant for binding to the operator site. In the case of the lac operon, the repressor protein is encoded by the i gene, which binds to the o operator site in order to prevent the transcription of the z, y, and a genes (the structural genes for β-galactosidase). There is also a promoter site, p, on the operon whose function is to direct the RNA polymerase to the proper transcription initiation site. All three structural genes, when transcribed, give a single mRNA that encodes β-galactosidase, the permease, and the transacetylase. Because this mRNA encodes more than one protein, it is called a polycistronic (or polygenic) transcript.

The lactose operon

The lac repressor protein in the absence of lactose binds to the operator and blocks transcription

The lac repressor (pictured bound to DNA) is a tetramer with amino- and carboxyl-terminal domains. The amino-terminal domain is the one that binds to the DNA while the carboxyl-terminal forms a separate structure. The two sub-units pictured consolidate to form the DNA-binding unit. When lactose is absent from the environment of the bacterium, the lac repressor binds to the operator DNA snugly and swiftly (4x10^6 times as powerfully to the operator as opposed to random sites on the genome). The binding of the repressor precludes RNA polymerase from transcribing the z, y, and a genes which are downstream from the promoter site and code for the three enzymes. The dissociation constant for the complex formed by the lac repressor and the operator is around 0.1 pM and the association rate constant is a whopping 10^10 M-1s-1. This suggests that the repressor diffuses along a DNA molecule to find the operator rather than via an encounter from an aqueous medium.

When it comes to the DNA-binding preference of the lac repressor, the level of specificity is so high that it can be called a nearly unique site within the genome of E. coli. When the dimers of the amino-terminal domain bind to the operator site, the dimers of the carboxyl-terminal site are able to attach to one of two sites within 500 bp of the primary operator site that approximate the operator’s sequence. Each monomer interacts with the bound DNA’s major groove via a helix-turn-helix unit.

Ligand binding can induce structural changes in regulatory proteins edit

Now let’s look at how the presence of lactose changes the behavior of the repressor as well as the expression of the operon. All operons have inducers—triggers that facilitate the expression of the genes within the operon—and the inducer of the lac operon is allolactose, a molecule of galactose and glucose with an α-1,6 linkage.

In the β-galactosidase reaction, allolactose is a side product and is produced at low levels when the levels of β-galactosidase are low in the bacterium. Additionally, though not a substrate of the enzyme, isopropylthiogalactoside (IPTG) is a powerful inducer of β-galactosidase expression.

In the lac operator, the way the inducer prompts gene expression is by inhibiting the lac repressor from binding to the operator. Its method of inhibition is by binding to the lac repressor itself thus immensely reducing the affinity of the repressor to bind to the operator DNA. The inducer binds to each monomer at the center of the large domain, causing conformational changes in the DNA-binding domain of the repressor. These changes drastically reduce the DNA-binding affinity of the repressor.

The operon is a common regulatory unit in prokaryotes edit

Numerous other gene regulation complexes within prokaryotes function analogously to the lac operon. An example of another network like this one is that which takes part in the synthesis of purine (and pyrimidine to a certain extent). These genes are repressed by the pur repressor, which is 31% identical to the lac repressor in sequence with a similar 3D structure. In this case, however, the pur repressor behaves opposite from the lac repressor: it blocks transcription by binding to a specific DNA site only when it is also bound to a small molecule called a corepressor (either guanine or hypoxanthine).

Transcription can be stimulated by proteins that contact RNA polymerase edit

While the previous examples of DNA-binding proteins all function by preventing the transcription of a DNA sequence until some condition in the environment is met, there are also examples of DNA-binding proteins that actually encourage transcription.

A good instance of this is the catabolite activator protein in E. coli. When the bacterium is grown in glucose it has very low amounts of catabolic enzymes whose function it is to metabolize other sugars. The genes that encode these enzymes are in fact inhibited by glucose, an effect known as catabolite repression. Glucose lowers the concentration of cAMP (cyclic AMP). When the concentration of cAMP is high, it stimulates the transcription of these catabolic enzymes made for breaking down other sugars. This is where the catabolite activator protein (CAP or CRP, cAMP receptor protein) comes into play. CAP, when bound to cAMP, will stimulate the transcription of arabinose and lactose-catabolizing genes. CAP, which binds only to a specific sequence of DNA, binds as a dimer to an inverted repeat at the position -61 relative to the start site for transcription, adjacent to where RNA polymerase binds (pictured).

This CAP-cAMP complex enhances transcription by about a factor of 50 by making the contact between RNA polymerase and CAP energetically favorable. There are multiple CAP binding sites within the E. coli genome, therefore increasing the concentration of cAMP in the bacterium’s environment will result in the formation of these CAP-cAMP complexes, thus resulting in the transcription of many genes coding for various catabolic enzymes.

Regulatory Circuits Can Result in Switching Between Patterns of Gene Expression edit

In investigating gene-regulatory networks and how they function, studies of bacterial viruses—especially bacteriophage λ—have been invaluvable. Bacteriophage λ is able to develop via either a lytic or lysogenic pathway. In the lytic pathway, transcription takes place for most of the genes in the viral genome which leads to the production of numerous virus particles (~100) and the eventual lysis of the bacterium. In the lysogenic pathway, the bacterial DNA incorporates the viral genome where most of the viral genes stay unexpressed; this allows for the viral genetic material to be carried in the replicate of the bacteria. There are two essential proteins plus a set of regulatory sequences within the viral genome that are the cause for the switch between the choice of pathways.

Lambda repressor regulates its own expression edit

λ repressor

λ repressor is one of these key regulatory proteins which promotes the transcription of the gene that encodes the repressor when levels of the repressor are low. When levels of the repressor are high, it blocks transcription of the gene. It is also a self-regulating protein. While the λ repressor binds to many sites in the λ phage genome, the one relevant here is the right operator, which includes 3 binding sites for the dimer of the λ repressor in addition to 2 promoters within an approximately 80 base pair region. The role the first promoter plays is driving the expression of the λ repressor gene, while the other promoter is responsible for driving the expression of a variety of other viral genes. The λ repressor binds to the first operating site with the most affinity; and when it is bound to this first operating site, the chances of a protein binding to the adjacent operating site increase 25 times. When the first and second operating sites have these complexes bound to them, the dimer of the λ repressor inhibits the transcription of the adjacent gene whose purpose is to encode the protein Cro (controller of repressor and others). The repressor dimer at the second operating site can interact with RNA polymerase so as to stimulate the transcription of the promoter which controls the transcription of the gene encoding the λ repressor. This is how the λ repressor facilitates its own production. λ repressor fusions can be used to study protein-protein interactions in E. coli. There are two different domains in λ repressor: the N-terminal (DNA binding activity) and the C-terminal domain (dimerization). In order to have an active repressor fusion, the C-terminal domain should be replaced with a Heterodimers domain and form a dimer or higher order oligomer. However, inactive repressor fusions cannot attach to the DNA sequences and affect the expression of phage or reporter.[1]

A circuit based on lambda repressor and Cro form a genetic switch edit

We can see in the above picture how the λ repressor blocks production of Cro by binding to the first operating site with the most affinity. Cro meanwhile blocks the production of the λ repressor by binding to the third operating site with the most affinity. This entire circuit is the deciding factor as to whether the lytic or lysogenic pathway will be followed: if λ repressor is high and Cro is low, the lysogenic path will be chosen; if Cro is high and the λ repressor is low, the lytic path will be chosen.

Many prokaryotic cells release chemical signals that regulate gene expression in other cells edit

Some prokaryotes are also known to undergo a process where they release chemicals called autoinducers into their medium (quorum sensing). These autoinducers, which are most of the time acyl homoserine lactones, are taken up by the surrounding cells. When the levels of these autoinducers reach a certain point, receptor proteins bind to them and activate the expression of several genes, including those that promote the synthesis of more autoinducers. This is a way for prokaryotes to interact with one another chemically to change their gene-expression patterns depending on how many other surrounding cells there are in their medium. Communities of prokaryotes that carry these mechanisms of quorum sensing out are collectively called a biofilm.

Gene Expression Can Be Controlled at Posttranscriptional Levels edit

Though most of gene expression regulation happens at the initiation of transcription, other steps of transcription are also possible targets for regulation.

Tryptophan Operon edit

Exploring the genes of the tryptophan operon (abbreviated as trp operon) in order to study the regulation of tryptophan synthesis shows two types of mutants. One type of mutant involves structural gene mutations and the other a regulatory mutant. The mutants that involve structural gene mutations are auxotrophic for tryptophan and need tryptophan to growth. To convert the precursor molecule chorismate to tryptophan, the trpE, trpD, trpC, trpB, and trpA genes codes for a polycistronic message and the mRNA will be translated to the enzyme that carries out the conversion.


The second type of mutants is able to constitutively synthesize the enzymes necessary for the synthesis of tryptophan. The trpR gene codes for the tryptophan repressor. The gene mapped in another quadrant of the E. coll chromosome compared to the trp operon. The trpR gene cannot regulate the synthesis of tryptophan efficiently. Studies on the dimeric trp repressor protein show that it does not function alone. The repressor must bind the last product of that metabolic pathway in order to regulate the synthesis of tryptophan. Thus, tryptophan is a corepressor for its own biosynthesis. This process is called feedback repression at the transcriptional level.

When the concentration of tryptophan is high enough, then the repressor binds to tryptophan to make a repressor-tryptophan complex. This complex will attach to the operator region of the trp operon and prevents RNA polymerase to bind and initiate transcription of the structural genes. Also, when the concentration of tryptophan is low in the cell, due to lack of tryptophan-complex RNA polymerase is able to bind to the gene and transcribe the structural genes. Therefore, tryptophan will be biosynthesized.[2]

Attenuation is a prokaryotic mechanism for regulating transcription through the modulation of nascent RNA secondary structure edit

While studying the tryptophan operon, Charles Yanofsky discovered another means of transcription regulation. The trp operon encodes 5 enzymes that convert chorismate into tryptophan, and upon examining the 5’ end of the trp mRNA he found there was a leader sequence consisting of 162 nucleotides that came before the initiation codon of the first enzyme. His next observation was that only the first 130 nucleotides were produced as a transcript when the levels of tryptophan were high, but when levels were low a 7000-nucleotide trp mRNA which included the entire leader sequence was produced. This mode of regulation is called attenuation, where transcription is cut off before any mRNA coding for the enzymes is produced.

Attenuation depends on the mRNA’s 5’ end features. The first part of the sequence codes for a leader peptide of 14 amino acids. The attenuator comes after the open reading frame for this peptide—it is an RNA region capable of forming a few alternate structures. Because transcription and translation are very closely coupled in bacteria, the translation of say the trp mRNA begins very soon after the synthesizing of the ribosome-binding site.

The structure of mRNA is altered by a ribosome, which is stalled by the absence of an animoacyl-tRNA necessary for the translation of the leader mRNA. This allows RNA polymerase to transcribe the operon past the attenuator site

References edit

  1. Leonardo Mariño-Ramírez, Lisa Campbell, and James C. Hu. Screening Peptide/Protein Libraries Fused to the λ Repressor DNA-Binding Domain in E. coli Cells. 2003; 205: 235–250.
  2. Arkady B. Khodursky, Brian J. Peter, Nicholas R. Cozzarelli, David Botstein. DNA microarray analysis of gene expression in response to physiological and genetic changes that affect tryptophan metabolism in Escherichia coli. 2000 October 24; 97(22): 12170–12175. Published online 2000 October 10.

The Control of Gene Expression in Eukaryotes

Chromatin are a combination of eukaryotic DNA and histones. The eukaryotic DNA binds tightly to the histones, which are basic proteins. Changes in the structure of chromatin are largely responsible for the regulation of gene expression. Other regulators of gene expression include interactions between proteins and translation.

In Eukaryotes, as compared to prokaryotes, gene regulation is a lot more complex. This is because the Eukaryotic genome is a lot larger and therefore encodes for a lot more proteins. There are also many different types of cells in eukaryotes, such as liver cells, pancreatic cells, and etc. The genes that are in these highly specialized cells have a huge difference in expression. Another reason for the complexity is that eukaryotic genes that encode proteisn are usually spread throughout the enormously large genome. The final reason is that eukaryotic transcription and translation are not coupled, and this negates some of the gene regulation mechanisms that prokarotes utilize.

Chromatin Structure

Chromatin is composed of units that repeat. Each of these units are composed of 200 base pairs of DNA, and two copies each of the four histones H2A, H2B, H3, and H4. This unit is called the histone octomer, and the repeating units are referred to as nucleosomes. The five main histones present in chromatin are H2A, H2B, H3, H4, and H1, but H1 is not part of the histone octomer. Each histone also has a flexible amino tail that have various lysine and arginine residues and extends beyond the core. These tails are very important because covalent modifications of them alter the DNA affinity of the histones. When chromatin is digested, it yields only 145 base pairs of DNA binding to the histone octomer, and this smaller unit is called the nucleosome core particle. The DNA that connects these nucleosome core particles in undigested chromatin is referred to as linker DNA. This is where H1 binds.

The three dimensional structure of the nucleosome is composed of eight histones arranged into a tetramer, and a pair of dimers. When the tetramer comes together with the dimers, they form a superhelical ramp that is left-handed, and DNA wraps around it forming a left-handed superhelix. The contact between the superhelical ramp and the DNA superhelix occurs primarily along the phosophodiester backbone and minor groove of the DNA. The winding of this DNA reduces its length by packing it together very tightly.

Chromatin Remodeling

The DNA adjacent to actively transcribed genes in chromatin are more sensitive to being cleaved, indicating that those sites contain less compact DNA. Additionally, some other sites, usually within proximity to the start of an actively transcribed gene, are also more sensitive to cleavage by nucleases. These sites are referred to as hypersensitive sites, and they either have fewer nucleosomes, or nucleosomes that are in an altered conformation. These hypersensitive sites specific to different cell types and are developmentally regulated. This indicates that a prerequisite for gene expression lies within chromatin.

Chromatin structure differs between active and inactive genes, and this indicates that some form of modification must be done to modify chromatin structure. Enhancers are DNA sequences that increase the activity of many promoters, even when they are thousands of base pairs away. Enhancers work by binding certain regulatory proteins, and they are only effective in the unspecific type of cell that express those regulatory proteins. These proteins may disrupt chromatin structure, exposing a gene and/or regulatory sites and thus influence transcription. This accounts for its ability to function at a distance from the gene being expressed.

DNA Modification

Gene expression can be inhibited by modifying DNA. This conclusion was drawn by studying sequences in the mammalian genomes. Lots of sequences in mammalian genomes have cytosines that are methylated at the C5 carbon. These cytosines have differing distribution throughout the genome depending on cell type.

Transcriptional Activation and Repression

Interactions between proteins mediate much transcriptional activation and repression. Eukaryotic transcription factors recruit proteins, which build large complexes that interact with and thus activate or repress transcription. This type of regulation is extremely advantageous because depending on the different proteins present in the cell, the regulation can have different effects. This is referred to as combinatorial control, and it is responsible for generating different types of cells.

Hormone receptors on the nucleus recruit various proteins to the transcription complex. These proteins are generally coactivators and corepressors. Coactivators are proteins that contain three repeated sequences within a central region of 200 amino acids. The repeated sequence is Leu-X-X-Leu-Leu, where X can be any amino acid, and they form short alpha helices that bind to the hormone receptors on the nucleus, inducing a change in conformation that enables the receptors to recruit the entire coactivator. Corepressors repress transcription. Sometimes repression can be done without binding a ligand, such as the receptors for retinoic acid and thyroid hormone. When unbound, the receptors bind to corepressors. The site in which the corepressor binds overlaps with the binding site for the coactivator and thus serves to repress transcription.

So What?

Tamoxifen and raloxifene are drugs used in the treatment of breast cancer. Tamoxifen inhibits the activation of gene expression by blocking coactivators from binding. This is important because cancer is such a prevalent disease in today's society, and by studying and understanding the mechanisms of gene activation and repression, new drugs can be produced to combat the disease by altering gene expression.

Source: Berg, Jeremy and Stryer, Lubert. Biochemistry: Fifth Edition. United States of America: W.H. Freeman and Company, 2002.

Alternative Splicing in Eukaryotes edit

Gene expression is the process that transfers genetic information from a gene made of DNA to a functional gene product made of RNA or protein. Genetic Information flows from DNA to RNA by the process of transcription, and then from RNA to protein by the process of translation. In order to ensure that the proper products are produced, gene expression is regulated at many different stages during and in between transcription and translation. In eukaryotes, the gene contains extra sequences that do not code for protein. In these organisms, transcription of DNA produces pre-mRNA, which must be spliced into mRNA that lacks these intervening sequences or introns, before translation begins. Splicing can be regulated so that different mRNAs can contain or lack regions called exons, in a process called alternative splicing. Alternative splicing allows more than one protein to be produced from a gene [1], and is an important regulatory step in determining which functional proteins are produced from gene expression. Splicing of pre-mRNA has been proven to be an important mechanism to controlling human development, and misregulation of splicing can lead to disease [2].

Diagram of alternative splicing

Mechanism of Alternative Splicing edit

Alternative splicing is a 2 step process carried out by the splicesome that ligates the 5’ splice site of an upstream exon to the 3’ splice site of a downstream exon. The splicesome is primarily composed of 5 small nuclear ribonuceloproteins (snRNP’s). snRNP’s carry out splicing through the recognition of 5’, 3’, and a branch point sequences in an intron.
Steps of Alternative Splicing:
1.) U1 snRNP recognizes GU of 5’ splice site, while U2 snRNP recognizes branch point bulged Adenosine.
2.) The A-complex is formed when U1 and U2 position the branch point near the 5’ splice site.
3.) The B-complex is formed when the tri-snRNP U4-U5-U6 bind to the A complex.
4.) The activated B-complex is formed when U1 and U4 leave, forming the catalytic splicesome.
5.) The C-complex is formed after the 1st step of splicing when the branch site adenosine attacks the 5’ splice site, creating an intron lariat.
6.) The 2nd step of splicing is carried out by the C-complex when the 5’ splice site attacks the 3’ splice site, resulting in the ligation of both exons. [3]

Regulation of Alternative Splicing by the rate of RNAPII Transcription edit

Mechanisms that influence splice site selection are important in regulating alternative splicing, and ensure production of the proper proteins at the right time. Many genes have been proven to be spliced co-transcriptionally [4], and the rate of RNA Polymerase II transcription has been suggested to be a regulatory mechanism of co-transcriptional Alternative splicing [5] [6]. Aebi and Weissman’s “first come, first served” model [7] shows how rate of transcription can influence the inclusion of internal exons. Slow recognition of the first intron or rapid recognition of the second intron creates a transcription dependent competition for 3' splice site selection. Slow transcription through the second intron could allow recognition of the first intron before the second is even synthesized, while fast transcription through the first two introns could increase the ability of the second intron to compete with the first intron for recognition. Formation of the “A Complex” (figure 2) is a step in alternative splicing which creates a commitment to splice site selection [8], and is completed by pairing of the U2 snRNP with the branch point [9]. This data shows control over the rate of transcription could be a very powerful tool in regulation of 3’ splice site selection.

One method eukaryotes use to regulate the rate of transcription is sequence based arrest sites located in DNA [10]. Using the thermodynamic theory of DNA-dependent transcriptional arrest, Artificial Arrest Sites (ARTAR sites) have been created and shown to affect rates of RNA Polymerase II transcription [11]. When placed downstream of both 3’ splice sites, the ARTAR site will have no effect on alternative splicing. If placed in a location in between the first and second branch points, the ARTAR site can increase inclusion by decreasing 3’ splice site competition.

References edit

  1. Douglas L. Black (2003) Mechanisms of Alternative Pre-Messeneger RNA Splicing. Annual Review of Biochemistry, 72: 291-336
  2. Mo Chen, James L. Manely (2009) Mechanisms of alternative splicing regulation: insights from molecular and genomic approaches. Nature, 10: 741-754
  3. Douglas L. Black (2003) Mechanisms of Alternative Pre-Messeneger RNA Splicing. Annual Review of Biochemistry, 72: 291-336
  4. Pandya-Jones A., Black DL (2009) Co-Transcriptional Splicing of constitutive and alternative exons. RNA, 10: 1896-908
  5. Kenneth James Howe, Caroline M. Kane, Manuel Ares Jr. (2003) Perturbation of transcription elongation influences the fidelity of internal exon inclusion in Saccharomyces Cerevisiae. RNA, 8: 993-1006
  6. Cramer P., Caceres J.F., Cazalla D., Kadener S., Muro A.F., Baralle F.E., Kornblihtt A.R. Coupling of transcription with alternative splicing: RNA pol II promoters modulate SF2/ASF and 9G8 effects on an exonic splicing enhancer (1999) Molecular Cell, 4 (2), pp. 251-258.
  7. Aebi, M. and Weissman, S.M. 1987. Precision and orderliness in splicing. Trends Genet. 3: 102-107
  8. Lim SR, Hertel KJ (2004) Commitment to splice site pairing coincides with A complex formation. Mol. Cell 15: 477–483.
  9. Qin Li, Ji-Ann Lee, Douglas L. Black (2007) Neuronal regulation of alternative pre-mRNA splicing. Nature Reviews Neuroscience 8: 819-831
  10. Spencer CA, Groudine M. Transcription elongation and eukaryotic gene regulation. Oncogene. 1990; 5:777–785.
  11. Dmitry Kulish, Kevin Struhl (2001) TFIIS Enhances Transcriptional Elongation through an Artificial Arrest Site In Vivo. Mol Cell Biol. 13: 4162-4168

Overview edit

Transition metals like zinc, iron, and copper are common components in a many different types of proteins. These metals are crucial for living; however, surplus amounts of these metals are detrimental to cell growth and viability. Luckily there are many mechanisms that help regulate this excess of metals when required. In response to these changes of metal levels, certain genes encodes transporting of metals and storage proteins that help maintain ideal levels of each metal. Deficiencies of these metals within cells can also cause health problems. Through many different types of complementary mechanisms, cellular metal homeostasis is achieved. Although not all transcriptional factors have been understood completely, they give researchers a sense of idea that could potentially explain the linkage between metal levels, health, and diseases.[1]

Metal levels can also be altered by different types of cancers, and even by neurodegenerative disorders such as Alzheimer’s and Parkinson’s diseases. Thus imbalanced metal levels can further cause severe implications when diagnosed with these illnesses. [1]

Different Types of Transition Metals edit

Zinc one of the vital transition metals in organisms, is important for the accurate folding of certain protein structures. It is the cofactor for hundreds of enzymes; however, in excess, zinc is very toxic to growth, and can wrongfully bind to unfitting locations in proteins and damage their function.[1]

Iron is another type of transition metal that is vital to life, acting as an electron acceptor or donor in physiological reactions. Like zinc, iron is also used as cofactors in enzymes required for oxygen transportation, DNA synthesis, ribosome biosynthesis, photosynthesis and many other functions. Like other transition metals, in excess, iron is very hazardous to growth and viability. Thus many organisms have evolved specialized mechanisms that regulate cellular iron levels. [1]

Found in a variety of eukaryotes, copper is a fundamental metal that is necessary for life in many organisms. Being a redox active metal, copper is a cofactor in many different proteins, including Cu/Zn superoxide dismutase 1 and cytochrome c oxidase. Like zinc and iron, when copper is in excess, it is highly toxic to cells and viability. Thus in order to maintain ideal level of copper, many eukaryotes developed transcription factors that regulate copper levels by controlling the genes that encodes for copper uptake and elimination.[1]

Metal Deficiencies edit

Zinc Deficiency edit

Changes in gene expression do not only occur in excess of zinc, but in deficiencies as well. In a variety of eukaryotic species, different sequence-specific DNA-binding factors have been found that are necessary for the variations in gene expression. Studying these zinc-regulated factors can provide a means to obtain certain types of proteins and different mechanisms that can be used to detect cellular zinc limitations in other organisms not known.[1]

Zap1 edit

Found in budding yeast Saccharomyces cerevisiae, the transcription factor Zap1, a zinc-responsive protein, can detect zinc levels. When zinc deficiency occurs, Zap1 can initiate the expression of about 80 genes, targeting genes that are require for zinc uptake and genes that can help survival during extreme zinc deficiencies. Zap1 can detect cellular zinc levels by a variety of domains. Zinc finger pair is a regulatory domain that overlaps with AD2, or activation domain 2. During zinc deficiencies, zinc binds to the zinc fingers within the AD2, which allows AD2 to undergo a conformational change causing exposures of residue that initiates the domain function. The zinc can rapidly switch from one of the zinc finger pair to the other, which allows the possibility of Zap1 to detect cellular zinc level. AD1, another activation domain within Zap1, is independently controlled by cellular zinc levels-activating gene expression in response to deficiencies. When AD1 is combined with AD2, it allows Zap1 to recruit more coactivators under precise stress situations. [1]

bZip19 and bZip23 edit

In another type of organism, more than one transcription factor are required for regulating genes during zinc deficiencies. Found in Arabidopsis thaliana, bZip19 and bZip23 activates the expression of genes required for zinc uptake.[1]

Iron Deficiency edit

Changes in gene expression do not only occur in excess of iron, they also occur in response to iron limitations as well and have been observed in many different eukaryotes such as green algae, fungi and plants. Most of the information that is known about gene transcription that has been affected by iron limitation is by the studying of yeasts. [1]

Aft1 and Aft2 edit

Found in Saccharomyces cerevisiae, Aft1 and Aft2, are transcription factors that responds to iron deficiencies by increasing the expression of about 40 genes. These genes can encode for certain proteins that require iron uptake, iron searching, or intracellular iron transport. Aft1 and Aft2 also regulate the expression of CTH1 and CTH2, genes which are iron-dependent, which help cells to conserve iron. Aft1 and Aft2 senses cellular iron deficiency by using a signal produced by the mitochondrial Fe-S cluster machinery. This signal however is still presently unknown, but studies revealed that Aft1 has the ability to sense these iron signals due to certain cytoplasmic proteins. [1]

Php4 edit

Found in fission yeast Schizosaccharomyces pombe, Php4, is another iron-responsive transcription factor that helps cells fight against iron deficiency. Similar to CTH1 and CTH2, Php4 controls the flux of iron through iron-depending pathways when iron is present. This is due to Php4 regulating genes that encodes for proteins that bind to iron or those found in metabolic pathways that needs iron. Transcription factor Grx4 is necessary to deactivate Php4 in high levels of iron concentration.[1]

Copper Deficiency edit

In many different eukaryotes such as yeast, plants, green algae, and fruit flies, cells can activate certain genes that encodes copper uptake when deficiency of copper occurs. Many of the transcription factors required for gene activation during copper limitation have been identified; however, how they function to detect deficiency have not been identified yet. Although only a few is shown here, there are often homologs of these transcription factors that provides the same role in a variety of other eukaryotes. Thus, there are many different proteins that have evolved using the equivalent copper responsive domain to detect multiple levels of coppers needed for homeostasis. [1]

Mac1 edit

Mac1 is a transcription factor found in the yeast Saccharomyces cerevisiae, becoming active when copper deficiency occurs and regulating the expression of genes required for copper uptake. In order for Mac1 to become activated, it requires both the Mac1 DNA-binding and transactivation domains. The transactivation domain comprise of cysteine-rich domains that are able to bind four copper ions. In copper excess, over binding of copper to this domain disables the Mac1 transcription factor. Mac1 DNA binding activity can become maximized with addition of Sod1, a rich copper-binding protein, during copper cellular deficiency. Thus Sod1 might have some unknown ability in copper sensing.[1]

Crr1 edit

In another species, Chlamydomonas reinhardtii, the green algae uses a different transcription factor that regulates copper limitation called Crr1. When copper levels are low, Crr1 can activate the expression of over 60 genes to achieve needed copper levels. Like Mac1, Crr1 also contains copper regulated domains, one is a cysteine-rich metal-responsive domain, and the other is SBP DNA binding domain. [1]

SPL7 edit

In another species, A. thaliana, copper levels are balanced by transcription factor SPL7, which is a homolog or descendant of the same ancestor, of Crr1. In order for copper homeostasis to become obtained, SPL7 expresses genes that are required for copper uptake and intracellular copper organization during copper deficiency. Different miRNAs can become activated by SPL7 that allows targeting of mRNAs that encodes for copper-binding proteins.[1]

Metal Excess edit

Zinc Excesses edit

In many multicellular organisms, there are distinct systems that detect zinc excess and deficiencies. MTF1 is a transcription factor that provides protection against zinc excess, which is very toxic to cells.[1]

MTF1 edit

MTF1 is a transcription factor found in mammals and fishes that helps protects cells from excess of zinc, activated when high concentration is present. It binds to metal-response elements which activates the target gene expression. Similar to Zap1, MTF1 senses cellular zinc excess by using regulatory zinc-finger domains. In contrast, the zinc fingers controls the binding of MTF1- only when zinc are in excess.[1]

Iron Excess edit

Studies obtained in a range of eukaryotes such as yeast, nematodes, flies and mammals, have noticed a variety of changes in gene expression due to excess of iron. Only in fungi however, have researchers identified transcription factors that are able to detect high levels of iron. Iron homeostasis is usually controlled at a post-transcription level within multicellular organisms.[1]

Yap5 edit

Found in Saccharomyces cerevisiae, transcription factor Yap5 protects cells from toxicity by expressing CCC1 activation when iron excess is present. Once Yap5 binds to the CCC1 prometer, CCC1 provides protection by transporting iron into an iron storage vacuole. CCC1 isn’t active unless high iron levels have been reached. Yap5 also comprises of cysteine-rich domains that are required for regulation of iron.[1]

Fep1 edit

This transcription factor is expressed when excess levels of iron have been reached. Found in S. pombe, Fep1 protects cells by stopping the expression of genes that acquires for iron when iron are at high concentration. Fep1 also regulates the expression of transcription factor Php4, controlling the flow of iron through metabolic pathways. Transcription factor Grx4 is required to deactivate Fep1 when iron levels are low, which is also required to deactivate Php4 when iron levels are high, which is likely the same signal that determine the switch of cells between both iron levels.[1]

Copper Excess edit

In order to protect eukaryotes species such as fungi and flies from copper toxicity, certain transcriptional factors are able to detect this high level of copper, and express regulatory mechanisms that can help protect cells. These factors can directly bind to copper and gene expressions are regulated. [1]

Ace1 edit

Found in Saccharomyces cerevisiae, Ace1 is a transcription factor that can bind directly to copper ions when copper is in excess. The binding to copper allows the activation of genes that help defend cells from toxicity that arises due to surplus of copper. A variety of other fungi have homologs of Ace1 that can also provide the same function by protecting against excess of copper. [1]

dMTF-1 edit

dMTF-1, a homolog of MTF1, regulates the expression of genes that are required for copper levels to become balanced when copper levels are in excess or in deficiency. dMTF-1 also have a cysteine-rich domain that functions in sensing copper toxicity by binding to four Cu+ ions.[1]

  1. a b c d e f g h i j k l m n o p q r s t u v w Link text

In cell biology, pluripotency is defined as "the potential of a cell to develop into more than one type of mature cell, depending on environment"[1]. So if a cell is pluripotent, it has the potential to transform itself into a lung cell, heart cell, etc. Pluripotency describes a cellular state of specific cells within the early embryo [2]. Stem cells represent a category of unspecialized cells that possess the unique capability of developing and maturing into a variety of specific types of cells with various functions.[3] Through the maturation and cell division processes, stem cells can specialize, grow, and replace unhealthy cells within living organisms. After cell replication, these cells have the ability to either mature and specialize in function, or remain an unspecialized stem cell, which preserves the cell’s potential to specialize as it is needed in the body. Not only are these cells able to specialize in function, but they are also capable of joining together to replace damaged tissues. The two types of stem cells scientists are currently studying are: embryonic stem cells and adult stem cells. In 1981, scientists first discovered methods to derive embryonic stem cells from mouse embryos, and in 1998, scientists made further developments and developed methods to derive and harvest stem cells from human embryos. Since stem cells are capable of self- replication, they offer great potential for treatment of diseases such as diabetes and heart disease. The use of these cells for treatment of certain diseases is called reparative medicine, and is currently a topic of much debate in present society and culture. Stem cells also offer the potential for use in screening new drugs as well as the study of how and why certain birth defects develop. Stem cell therapy offers a great deal of insight into medicine as well as human growth and development, and research is currently underway in order to find methods to fully implement all the advancements these cells could provide to modern medicine.

Stem cells are characterized by their ability to differentiate into a diverse range of specialized cells and to grow indefinitely. They have two properties:

  • Self-renewal: The ability to undergo cell division indefinitely while maintaining their undifferentiated state. Stem cells are capable of doing so due to having active telomerase.
  • Potency: The ability to differentiate into specialized cell types in response to a signal. Skin cells, heart cells, and neurons all have the same genome, but are all different cells.[4] This is due to differentiation and different RNA/protein combinations.

Stem cells are often classified into one of two categories: embryonic stem cells, or adult stem cells.

Totipotency edit

Totipotency is defined as a cell's ability to transform into all cell types of the organism[5]. Both oocyte (the female germ cell involved in reproduction. This is an immature ovum) and zygote (this is the initial cell created after tow gamete cells are merged by means of sexual reproduction) do not possess the potential to be totipotent. However, the fertilized egg becomes equipotent blastomeres from its specialized cell type with a limited fate. Blastomeres are a type of cell created by cleavage of the zygote after fertilization [6].

What researchers have found through a series of transplantation experiments 1. Maternal proteins and RNAs are taken into the oocyte and deposited there 2. Those maternal proteins and RNAs have the capability to reset the epigentic state of somatic chromatin 3. They also serve to turn the oocyte into totipotent blastomeres after fertilization 4. So far, it has not been achieved to make totipotent cell lines in vitro. 5. This may be impossible due to the fact that in vivo, that is within living body, the transient nature of the totipotent is extremely different. The pre-embryonic stage of embryonic development offers blastomeres that are not actually self-renewin but they are generated by means of cleavage

Primordial Pluripotency edit

Slowly, a restriction in development potency become apparent as cleavage of the early embryo approaches to the 16-cell stage. Then the cells undergo two different lineages, the trophoblast lineage and the inner cell mass. The question is how the cell differentiation begins. Differentiation begins as blastomeres flatten and as they strengthen cell-to-cell contact. This process is called compaction.

Embryonic Stem Cells edit

Specialization of Embryonic Stem Cells

Embryonic stem cells are cells that are derived from embryos that have typically been harvested from eggs through in-vitro fertilization.[7] Embryonic stem cells are known as pluripotent, or having the ability to differentiate into every cell line within the organism. The embryos are 3–5 days old and consist of collections of hollow balls of cells that are called blastocysts.

In order to produce these blastocytsts, the embryonic stem cells must be harvested. The process of growing these stem cells is called cell culture, and is performed in the laboratory. The inner cell mass of human embryonic stem cells are placed onto a culture dish that has been coated with a medium that provides an adhesive surface to which the inner cell masses can stick. This medium is called a feeder layer, and is oftentimes composed of mouse skin cells. The human embryonic stem cells are then allowed to divide, spread, and attach to the medium of the culture dish in order to create a colony of embryonic stem cells. If the stem cell colony grows, divides and survives, the cells are plated onto new, clean culture dishes many times for the duration of many months in order to yield millions of embryonic stem cells, which is called a stem cell line. A sign that a stem cell line has developed successfully is if the cells have remained unspecialized for the duration of at least six months. If this is the case, the stem cell line can then be used for further testing and experimentation.

Throughout the harvesting process the embryonic stem cell colony must undergo a process called characterization, which involves a series of tests performed by scientists to see whether or not the cells have in fact developed correctly. One such test involves harvesting and examining the stem cell colony for many months in order to verify that the cells are healthy and capable of successful renewal. These cells are also analyzed to make sure they remain unspecialized. A way to test whether or not the cells have specialized is to test for the presence of transcription factors that are produced by unspecialized cells. Two examples of these transcription factors are Nanog and Oct4. The presence of these transcription factors indicate the cells have remained unspecialized and that they are capable of self replication and renewal.

Another test performed on embryonic stem cell cultures involves freezing, thawing and re-plating the cells to see whether or not they are capable of self renewal and re-growth.

An important test to examine whether or not the cells are functioning properly involves injecting a collection of embryonic stem cells into a mouse with a suppressed immune system followed by tests that indicate the formation of teratomas, which are benign tumors. If the injected stem cells successfully form a teratoma, scientists can be confident that the cells are capable of growth and differentiation, since teratomas contain various specialized cells.

Embryonic stem cells (ESC) are harvested from a blastocyst, which is an early stage in embryonic development(approximately four to five days in humans) that consists of 100-150 cells. ESC give rise to all three primary germ layers during development: ectoderm, endoderm, and mesoderm; which means they can develop into more than 200 specialized cell types. They can grow indefinitely under the right conditions due having active telomerase. Telomerase is inactive in adult cells, and as a result, cannot grow indefinitely.

Embryonic stem cells have proven to be usable in different situations:

  • Embryonic development: Understanding ESC aids scientists in further understanding embryonic development.
  • Drug testing: Certain cells cannot be cultured because they will die if removed from an organ or tissue. For example, in designing drugs for the heart, scientists cannot culture heart cells because once the heart cells are removed, they die. However, ESC can be cultured, and the ones that are differentiated into heart cells can be sustained in a culture to be used to test the drug.
  • Regenerative Medicine: This is not something ESC are currently being used for, but something that scientists are striving towards. The theory, or goal, is to be able to remove the nuclei in an embryonic stem cell, insert a patient's DNA into the cell, and have it differentiate according to the patient's needs. For example, Parkinson's disease is a degenerative disease of the central nervous system, and in theory, by adding back new neurons, it should be able to cure Parkinson's.
  • Paralysis: In addition, those who suffer from paralysis and unmovable body parts could regenerate body motor control through the study of stem cell research.
  • Leukemia: Patients suffering from Leukemia who may be in need of bone marrow transplant or cord blood transfusion can benefit from these stem cell researches. These products could be reproduced without having the need to constantly find a matchable donor. Although this may not treat the disease, it can diminish the symptoms greatly.

Specialization edit

The purpose of harvesting embryonic stem cells is to create a colony of self replicating cells that have the potential to specialize into specific cells of interest that can be used to treat diseases.While they are being grown, the cells remain unspecialized but once they are allowed to clump together under certain specific conditions, they begin to form various specialized cells and tissues. The challenge scientists are currently facing is figuring out how to control the specialization to create specific cells of interest as opposed to spontaneous specialization that can result in the formation of various cells that carry unwanted specific functions. One such way to control specialization is by subjecting the colonies of embryonic stem cells to changes in chemical composition within the culture medium, changing the surface of the culture dish, or inserting specific genes into the culture to change the composition of the harvested cells. By transplanting the successfully specialized embryonic stem cells into humans, scientists can formulate treatments for certain diseases such as Parkinson’s Disease, diabetes, spinal cord injury, heart disease as well as vision and hearing loss.

Adipogenesis edit

Adipocytes are cells specialized for energy storage and so when the balance of energy intake versus expenditure is shifted so that more energy is taken in than is burned, the process of adipogenesis begins. This process involves adipocyte hyperplasia, the excessive recruitment of stem cells to become adipocytes, thereby increasing the number of adipocytes. In order to accomplish this, pluripotent mesenchymal stem cells (MSCs) become preadipocytes then are further developed into mature adipocytes.

The pluripotent MSCs located in the vascular stroma of adipose tissue and bone marrow first become preadipocytes, a process initiated by bone morphogenic proteins BMP4 or BMP2, then after several rounds of mitotic clonal expansion, they become adipocytes. While this process is activated by the BMP factors, other factors such as Hh provide inhibitory signals if the process becomes unnecessary[8].

Regulation and Signaling of Stem Cells edit

For stem cells, transcription factors are important to their functioning and can be categorized into three central groups; 1. ones affecting development and its hierarchy (Developmental Core Module), 2. ones controlling cell growth and homeostasis (Cell Growth Module), and 3. ones that control signaling pathways of the other two groups (Signaling Module). These three groups mostly function independently, though there is some interaction present.

Developmental Core Module edit

The best understood of transcription factors controlling cell pluripotency is Oct4 which is an octamer class protein that specifically recognizes the sequence ATGCAAAT.[9] Oct4 is unique apart from the other transcription factors in that it is crucial for epigenetic reprogramming (differentiation of cells during development). If it is inhibited or silenced in any way, ESCs will instantly stop self-renewing and differentiation into trophoblast-like cells. Oct4 will interact with many cofactors in the form of transcription factors. These cofactors tend to interact with DNA and include Sall4, Sp1, Hdac2, Nanog, Dax1, Nac1, Tcfp2l1, and Essrb. Their interactions do not form one protein nexus but multiple complexes all with different composition of proteins. This differing of composition and complexes may be the reason for different states of pluripotency.

Sox2 is another transcription factor involved in development and cell pluripotency. It has an expression pattern that is, to some extent, similar to Oct4. However, when inhibited or silenced, this will mainly affect the later stages of embryogenesis. Sox2 silencing will only affect a small portion its target genes and will lead to trophoblast-like cells. This is mainly because Sox2 loss can affect the amount of Oct4 levels in the cells which is the main reason for the creation of the trophoblast-like cells.

Nanog is also a member in the core developmental module and is a homeodomain-containing DNA-binding factor that is not associated with the normal ones. Nanog expression alone is able to help keep cell pluripotency in extreme conditions where factors are missing. It has been shown in studies to have a role of starting cell pluripotency rather than maintaining it. Nanog has cofactor proteins it interacts with which includes Smad1, Small3, Nr0b1, Nac1, Essrb, Zfp281, Hdac2, and Sp1.

Cell Growth Module edit

This is the gene network that controls and maintains the growth and proliferation of pluripotent stem cells.

The proto-oncogene c-Myc is one member of this group is associated with activating transcription and opening chromatin. Its expression allows ESCs to self-renew even in extreme conditions. If it is inhibited ESCs will shut off their self-renewing mechanism. c-Myc also controls transcription of genes from multiple unconnecting functions such as cellular metabolism, control of cell cycle, and manufacture of protein. It has an inhibition factor also and will prevent the expression of Gata6 which is a prodifferentiation factor.[9] It also has been found to overlap with functions from genes of the developmental module such as Tip60 and p400. It has also seen to function in some cases of cancer growth. c-Myc expression is regulated by the transcription factor Ronin (Thap11) which is part of a family of proteins containing an N-terminal THAP domain. It is well conserved between mice and humans which means its sequence and target sequence is very similar for both mammals. Ronin inhibition will led to cell apoptosis while overexpression will lead to an abundance of mESCs.

Signaling Module edit

There are several signaling pathways that will directly promote self-renewal and/or preserve pluripotency by blocking or signaling normal differentiation prompts.

Lif (Leukemia inhibitory factor) stimulates the self-renewal of mESCs. It does not directly increase the growth rate of mESCs but rather increases the probability that the cells with self-renew rather than differentiate. The Lif-signaling effect is controlled by a heterodimeric complex that is made of a Lif receptor (LifR) chain and the protein gp130. It has been shown to signal three pathways which are the Jak/Stat3, the PI3K-Akt, and Mapk pathways.[9] Of the three, only the Jak/Stat3 pathway completely depends on Lif for its signal. The other two pathways require multiple signals from multiple sources in other to start the process. Signaling of the Jak/Stat3 pathway and PI3K pathways will promote expression of Klf4 and Tcf3 which lead to the creation of greater transcription of Sox2 and Nanog which as stated earlier are involved in differentiation of stem cells and development of the body. For the Mapk pathway, its signaling will lead to less amounts of Tbx3 which will cause a decrease of transcriptional activity. FgfR1 (Fibroblast growth factor signaling) is the most ample receptor of ESCs in the body. It is involved in signaling the Jak/Stat, phophoinositide phospholipase C-y, PI3K, and Erk pathways. A similar protein, Fgf2 is important for the maintenance of hESC. For example, in mouse feeder cell cultures a hot amount of Fgf2 is required to maintain its self-renewal and prevent differentiation.

Wnt signaling is very important during embryonic development and controls differentiation of stem cells. Its can induce expression of mesodermal and endodermal markers that, for mESCs, includes Barchyury, Flk1, Foxa2, Lxh1, and Afp. Wnt signaling also maintains cell stem pluripotency by controlling the expression of Oct4, Sox2, and Nanog which are all transcription complexes involved in maintaining pluripotency and development.[9]

Pluripotency-Associated Chromatin Structure edit

It should be noted that every cell has a unique chromatin structure. Chromatin is the combination of DNA and proteins that make up the contents of the nucleus of a cell[10]. In pluripotent cells, heterochromatic-associated foci seems to be more spaciously distributed. The associated histones are usually hyperacetylated. There exists chromatin-modifying enzymes that play a role with the transcriptional modules. They do so by controlling genes that encode key enzymes capable of covalently modifying proteins[11]. For instance, the transcription of the H3K9 methyltransferase SetDB1 is controlled by Oct4. It is known that histone labels characteristic of this enzyme. When this happens, the following things occur; 1. Polycomb group proteins are introduced 2. Polycomb group proteins trigger subsequent events which involve methylation of H3K27 residues 3. Then further additional chromatin-associated modifications are enabled.

Sources of Embryonic Stem Cells edit

1. In Vitro Fertilization edit

The largest potential source of blastocysts for stem cell research is from in vitro fertilization (IVF) clinics. The process of IVF requires the retrieval of a woman's eggs via a surgical procedure after undergoing an intensive regimen of "fertility drugs," which stimulate her ovaries to produce multiple mature eggs.

When IVF is used for reproductive purposes, doctors typically fertilize all of the donated eggs in order to maximize their chance of producing a viable blastocyst that can be implanted in the womb. Because not all the fertilized eggs are implanted, this has resulted in a large bank of "excess" blastocysts that are currently stored in freezers around the country. The blastocysts stored in IVF clinics could prove to be a major source of embryonic stem cells for use in medical research. However, because most of these blastocysts were created before the advent of stem cell research, most donors were not asked for their permission to use these left-over blastocysts for research.

The IVF technique could potentially also be used to produce blastocysts specifically for research purposes. This would facilitate the isolation of stem cells with specific genetic traits necessary for the study of particular diseases. For example, it may be possible to study the origins of an inherited disease like cystic fibrosis using stem cells made from egg and sperm donors who have this disease. The creation of stem cells specifically for research using IVF is, however, ethically problematic for some people because it involves intentionally creating a blastocyst that will never develop into a human being.[12]

2. Nuclear Transfer edit

The process called nuclear transfer offers another potential way to produce embryonic stem cells. In animals, nuclear transfer has been accomplished by inserting the nucleus of an already differentiated adult cell-for example, a skin cell-into a donated egg that has had its nucleus removed. This egg, which now contains the genetic material of the skin cell, is then stimulated to form a blastocyst from which embryonic stem cells can be derived. The stem cells that are created in this way are therefore copies or "clones" of the original adult cell because their nuclear DNA matches that of the adult cell.As of the summer of 2006, nuclear transfer has not been successful in the production of human embryonic stem cells, but progress in animal research suggests that scientists may be able to use this technique to develop human stem cells in the future.

Scientists believe that if they are able to use nuclear transfer to derive human stem cells, it could allow them to study the development and progression of specific diseases by creating stem cells containing the genes responsible for certain disorders. In the future, scientists may also be able to create "personalized" stem cells that contain only the DNA of a specific patient. The embryonic stem cells created by nuclear transfer would be genetically matched to a person needing a transplant, making it far less likely that the patient's body would reject the new cells than it would be with traditional tissue transplant procedures.

Although using nuclear transfer to produce stem cells is not the same as reproductive cloning, some are concerned about the potential misapplication of the technique for reproductive cloning purposes. Other ethical considerations include egg donation, which requires informed consent, and the possible destruction of blastocysts.[12]

Adult Stem Cells edit

Adult stem cells are known as mesenchymal stem cells. Just like embryonic stem cells, adult stem cells are unspecialized cells capable of renewal and specialization, but unlike embryonic stem cells, adult stem cells are typically found in tissues surrounded by other specialized cells. This makes the stem cell only multipotent; it can only differentiate into certain cell lines as opposed to pluripotent embryonic stem cells which can differentiate into almost any cell line. The role of adult stem cells is to replenish damaged cells within tissues and organs when needed. Adult blood-forming stem cells from bone marrow have been used for transplants, and current research is underway to determine whether or not differentiation of stem cells found in the brain and heart can be controlled and used for various other forms of transplantation.

Adult stem cells are found in areas called “stem cell niches” in various organs and tissues. These cells remain dormant until there is a need for them, which could include a requirement for new cells to replace damaged tissue due to injury or disease. Like embryonic stem cells, scientists are developing methods to extract these adult stem cells, harvest them in colonies, and manipulate them to create colonies of specialized cells that can be used for treatments of certain diseases. The challenge arises because once extracted from the human body, these cells have limited capacities to grow and reproduce.

A way scientists identify adult stem cells is first by extracting cells from a living organism. Next, the cells are labeled in a cell culture and transplanted back into a different organism. If the cells reproduce in the organism, scientists can verify the presence of properly functioning stem cells. Scientists have been conducting research to identify various differentiation pathways, which are the processes by which adult stem cells differentiate and convert into highly specialized cells with specific functions.

Different types of adult stem cells include the following:

  • Hematopoietic stem cells: develop into bone cells, cartilage cells, fat cells
  • Neural stem cells: develop into neurons, astrocytes and oligodendrocytes
  • Skin stem cells: develop into epidermal stem cells and hair follicles

Adult stem cells are found in developed organisms and have the ability to divide and create another cell like itself, or create a cell more differentiated than itself.

They have a few differences from embryonic stem cells, which include:

  • They cannot grow indefinitely due to having inactive telomerase. Some can actually only double once in their cellular life span.
  • Adult stem cells have a limited ability do differentiate, unlike ESC.
  • They exist in much smaller quantities than ESC do. There exists about 1 adult stem cell for every 10,000-15,000 cells in an adult human.
  • There also exists less ethical issues regarding adult stem cells because they are extracted from adults who are able to make their own decisions and give consent.

Potential exists for adult stem cells and regenerative properties, such as:

  • Healing organs: In the event of a heart attack, heart cells die and are replaced by scar tissue. Adult stem cells have been used to partially heal the scar tissue in animals by removing the damaged heart tissue, culturing adult stem cells in a petri dish, and putting them back in the damaged heart.
  • Organ regeneration: In 1999, Dr. Anthony Atala, Urologist and Director of the Wake Forest Institute for Regenerative Medicine, was studying patients with a bladder defect. He took a small piece of bladder, removed the stem cells, and actually grew it into a new bladder. He discovered that he could stick them back in the original patient, and seven years later, the patients were either better, or like normal. The bladder he was able to create was functional enough to keep the patients healthy and alive.

CD34+ Cells edit

CD34+ cells contain the protein CD34. CD34 protein is a glycoprotein found on human cell surfaces and functions as an adhesive for cells to interact. It could also have a function in attaching stem cells to bone marrow or connective tissue cells. The protein is expressed early in hematopoietic cells. Another function is that it is needed for T cells to enter lymph nodes. CD34+ cells are commonly found in as hematopoietic stem cells in umbilical cords and bone marrow.

Uses edit

To isolate CD34+ cells from blood samples, immunomagnetic or immunofluorescent techniques are used. These hematopoietic stem cells can be purified for research or clinical use in bone marrow transplant, by using antibodies. These cells can differentiate into different blood cells. CD34+ cells can be injected into patients to treat diseases such as Liver Cirrhosis, Peripheral Vascular disease, and spinal cord injury.

Induced Pluripotent Stem Cells edit

Induced pluripotent stem cells or iPSCs are adult somatic cells that have been reprogrammed to a state similar to embryonic stem cells. Although iPSCs are theoretically pluripotent cells, it remains unknown if they differ at all from embryonic stem cells. Induced pluripotent stem cells were first produced in 2006 from mouse cells and human cells followed in 2007.

Production of iPSCs edit

iPSCs are produced through a transfection which is process of introducing nucleic acids into a cell.[13] In the case of iPSCs, this done through retroviruses. Other methods do exist in the production of iPSCs such as the use of adenoviruses and plasmids. Adenoviruses differ from retroviruses because it does not incorporate its own genetic information into the adult cell. Adenoviruses are used to transport the necessary transcription factors into the DNA of the adult cells. Adenoviruses present some benefits such as the fact that they avoid any potential for mutagenesis that may stem from the insertion as well as the fact that adenoviruses only need to be present for a small period of time to reprogram the adult cell. Plasmids can also be used to reprogram the adult cell and in this method, two plasmids are required to carry the necessary transcription factors. The main benefit of using plasmids is the simple fact that viruses do not have to used. Two main problems exist for the use of plasmids though and they are the fact that plasmids demonstrate low efficiency as well as the fact that plasmids have a continued risk of insertional mutagenesis and use cancer-promoting genes during reprogramming.

Through Cell Fusion edit

IPSCs have been created successfully by fusing EGCs (embryotic germ cells) or ESCs (embryotic stem cells) with somatic cells.[9] Somatic cell nuclei can be reprogrammed by EGCs and mESCs after they are fused with thymic lymphocytes. After reprogramming, the procedure leads to tetraploid cells that resemble EGCs where the female cells have their inactive X chromosomes became active again. These tetraploids can then be injected into blastocyst to form chimeric embryos. Specifically for humans, this can, in the future, be used for both bone marrow cells and brains cells in the central nervous system that have been isolated. These cells can then be fused with mESCs in culture to create artificial cells that are similar to real ones in morphology and characteristics.

Through Cell Reprogramming edit

iPSCs can be also be obtained through reprogramming somatic cells (body cells of an organism) into pluripotent stem cells.[9] This procedure is performed by the use of transcription factors which are proteins that bind to specific DNA sequences. The four transcription factors that are used in mouse fibroblasts via retroviral infection are Oct4, Sox2, Klf4, and c-Myc. For humans, different combinations of transcription factors have been used which includes Nanog, Lin28, or Nr5a2. The exogenous expression of the transcription factors is followed by selection in a mESC (mouse embryotic stem cell) agent to finally lead to the iPSCs. The resulting iPSCs have characteristics of normal ESCs (embryotic stem cells) such as reactivation of both X chromosomes in female cells, expression of endogenous pluripotency indicators, and the ability to create chimeric animals. The molecular events are still poorly understood, but how reprogramming occurs can be investigated. During reprogramming, epigenetic marks are expansively altered. Studies have shown that iPSCs clones to be impossible to tell apart from ESCs though there are still small differences that need to be tested before they can be used in regenerative medicine.[9]

The Clinical Future of iPSCs edit

Induced pluripotent stem cells promise the almost infinite possibilities of embryonic stem cells without the nagging moral implications of the destruction of human embryos, and for this reason offers a promising place for research for regenerative medicine. iPSCs also hold the promise of other benefits over other types of stem cells. The main one being the fact that iPSCs can be developed from a patients own cells which mean that any treatments derived from the iPSCs should avoid any immunogenic responses. Even with the enormous possibilities of iPSCs in a clinical setting, a major concern hovers over the future which is the fact that iPSCs have an increased tendency to form tumors. From experiments, it was discovered that iPSCs injected into mice formed teratomas which are tumors with tissue or organ components inside. Moving toward the future, research is focused on not only coming up with potential treatments using iPSCs but on producing methods with higher efficiency as well as decreased occurrences of tumors using recombinant proteins.

Uses of Stem Cells edit

Both embryonic and adult stem cells have the potential to provide both treatments for various diseases as well as insight into human growth and development. One specific area of stem cell therapy is the use of these cells to treat people with type I diabetes. Type I diabetics are unable to produce insulin in the pancreas. Current research is underway to determine whether or not embryonic stem cells can be selectively specialized to give rise to insulin producing colonies that can be transplanted into diabetic patients. In order for such a procedure to be successful, stem cells must be capable of reproducing and generating enough of the tissue/cells of interest. They must also be able to survive in the recipient host, function properly, and maintain the capability to reproduce, while leaving the recipient unharmed. All of these requirements must be met without the recipient rejecting the cells, which is a challenge researchers are presently trying to overcome.

Human stem cells could also be used to test new drugs. New medications could be tested for safety on differentiated cells generated from human pluripotent cell lines. Cancer cell lines for example are used to screen potential anti-tumor drugs. Using stem cells scientists are also developing new ways to grow brain cells in the laboratory that could be used to treat patients with Parkinsons disease. Among the various approaches one is to grow stem cells into brain cells and transplant them into patients. If stem cells can be cultivated to become dopamine-producing nerve cells, researchers believe that they could replace the lost cells.

By using stem cells one could observe the specailization of specific cells such as nerve and muscle cells. By observing this process some of the most serious medical conditions, such as cancer and birth defects, are due to problems that occur somewhere in this process of specialization. Another potential application of stem cells is making cells and tissues for medical therapies.

Cancer cells have the ability to divide indefinitely and spread to different parts of the body during metastasis. Embryonic stem cells can self-renew and, through differentiation to somatic cells, provide the building blocks of the human body. Embryonic stem cells offer tremendous opportunities for regenerative medicine and serve as an excellent model system to study early human development.

Controversy Surrounding Research edit

There is much ethical controversy regarding embryonic stem cell research. The number one being how ESC are harvested because once ESC are retrieved, the blastocyst dies. There is no way to harvest them without destroying the embryo. This brings to the table the question of when does life begin? There are many different beliefs as to when life begins, whether it be scientific, religious, etc., and so it is difficult to set a definition that everyone can agree upon.

Most of the blastocysts that are used for stem cell research come over from excess invitro fertilization. People who have fertility issues and decide to have invitro fertilization. This is where sperm and an egg are combined in a test tube, which results with a number of embryos, some of which are implanted in the mother. The left over embryos are then frozen for a variety of reasons; whether the first implantation doesn't take, or the couple decides to have a second child later on. Some decide to discard the embryos, but a vast majority are left frozen in labs. Another controversial question that arises is that whether it is alright to use the frozen embryos for ESC.

Political controversy in research arises as well:

  • late '90s: The first embryonic stem cell line was created.
  • 2001: Bush outlaws the creation and studying of more embryonic stem cell lines with the use of federal money. There were 21 distinct ESC lines at the time.
  • 2009: Obama creates the National Institute of Health (NIH) panel that will approve whether or not federal funding can be used to study certain stem cell lines. Established guidelines state that the stem cells must come from embryos that were created through invitro fertilization but were frozen and not going to be used anymore. Parents must provide consent for their frozen embryos to be used for stem cell research, and once approved by the NIH federal money can be used to study them. However, federal money cannot be used to create new stem cell lines. They must be created through private funding, and once they are created, federal funding can be used to study it.

An issue of the overall safety of the use of stem cells exists as well. Researchers do not necessarily know what will happen when stem cells are inserted into a person, they know what they WANT to happen, but cannot guarantee what will. Scientists have yet to be able to precisely control what cells an embryonic stem cell will differentiate into.

References edit

  2. “Pluripotency and Nuclear Reprogramming” From Annual Review of Biochemistry Vol. 81 737-765, by Marion Dejosez and Thomas P. Zwakav
  4. Takahasi, Kazutoshi, and Shinya Yamanaka. "Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors." Cell 126 (2006): 663-76. Invalid <ref> tag; name "tak" defined multiple times with different content
  5. “Pluripotency and Nuclear Reprogramming” From Annual Review of Biochemistry Vol. 81 737-765, by Marion Dejosez and Thomas P. Zwaka
  6. ^ Blastomere Encyclopædia Britannica. Encyclopædia Britannica Online. Encyclopædia Britannica Inc., 2012. Web. 06 Feb. 2012.
  7. Oliver Dreesen and Ali Brivanlou.Stem Cell Reviews and Reports,Humana Press Inc.
  9. a b c d e f g Dejosez, Marion, and Thomas P. Zwaka. "Pluripotency and Nuclear Reprogramming." Annual Review of Biochemistry 81 (2012): 737-65. Annual Reviews. Web. 6 Dec. 2012.
  11. “Pluripotency and Nuclear Reprogramming” From Annual Review of Biochemistry Vol. 81 737-765, by Marion Dejosez and Thomas P. Zwaka
  12. a b
  13. Bellin, Milena, Maria Marchetto, Fred Gage, and Christine Mummery. "Induced Pluripotent Stem Cells: The New Patient?" Nature Reviews Molecular Cell Biology 13 (2012): 713-26.

Overview edit

There is only one type of cell that is completely generic—its gene expression is tuned so broadly that it has unlimited career potential to become any kind of cell in the body. These undifferentiated cells exist a few days after conception and form the blastula, consisting of three layers, the ectoderm, endoderm, and mesoderm and about 100 cells. Embyonic stem cells have an almost unlimited capacity to divide due to high levels of telomerase that prevents telomare degradation in aging cells. Shortly after, a wave of growth hormones such as Testosterone and Sonic Hedgehog, induce the blastocyst to change from a spherical structure and begin to take on the rough morphology that the animal has.

Uses edit

Due to their almost unlimited capacity to divide and their potential to divide into any kind of cell in the body, embryonic stem cells are being looked at as therapies for many different types of neurodegenerative therapies such Parkinson's, Alzheimer's, and Huntingtons in which the neurons in an aging brain or body die and are not replaced. by attempting to induce stem cells to differentiate into these types of neurons and repopulate the affected regions; the progress of the disease can be halted or even reversed.

Embronic stem cells are also being considered for organ transplantations. If Embryonic stem cells are saved at birth from the placenta and umbilical cord. These cells can theoretically then be used to grow organs with the same blood type and identifying proteins in them, eliminating the need to wait for an organ donor which reduced the organ shortfall already present, and ensuring that the organ is a perfect match with the recipient's body, eliminating the chance of rejection and the need for anti rejection drugs.

Ethical Issues edit

Because embryonic stem cells are so few, numbering only a hundred or so per embryo, they are very expensive and difficult to harvest and maintain. This process inevitably destroys the developing embryo leading to charged political debate versus the cost of sacrificing a potential life versus the enormous benefits these types of cells could bring.[1]

Application edit

Although researchers have been studying stem cells from mouse embryos for more than 20 years, only recently have they been able to isolate stem cells from human embryos and grow them in a laboratory. In 1998, James A. Thomson of the University of Wisconsin, Madison, became the first scientist to do this. He is now at the forefront of stem cell research, searching for answers to the most basic questions about what makes these remarkable cells so versatile. Although scientists envision many possible future uses of stem cells for treating Parkinson’s disease, heart disease, and many other disorders affected by damaged or dying cells, Thomson predicts that the earliest fruits of stem cell research will be the development of powerful model systems for finding and testing new medicines, as well as for unlocking the deepest secrets of what keeps us healthy and makes us sick.[1]

References edit

  1. a b U.S. Department of Health and Human Services. Inside the Cell. September 2005.<>.

Overview edit

Long after our embryonic stem cells have differentiated, we all still harbor other types of multitalented cells, called adult stem cells. These cells are found throughout the body, including in bone marrow, brain, muscle, skin, and liver. They are a source of new cells that replace tissue damaged by disease, injury, or age.

Researchers believe that adult stem cells lie dormant and largely undifferentiated until the body sends signals that they are needed. Then selected cells morph into just the type of cells required. Like embryonic stem cells, adult stem cells have the capacity to make identical copies of themselves, a property known as self-renewal. But they differ from embryonic stem cells in a few important ways. For one, adult stem cells are quite rare. In addition, adult stem cells appear to be slightly more “educated” than their embryonic predecessors, and as such, they do not appear to be quite as flexible in their fate.


Application edit

However, adult stem cells already play a key role in therapies for certain cancers of the blood, such as lymphoma and leukemia. Doctors can isolate from a patient’s blood the stem cells that will mature into immune cells and can grow these to maturity in a laboratory. After the patient undergoes high-dose chemotherapy, doctors can transplant the new infection-fighting white blood cells back into the patient, helping to replace those wiped out by the treatment. Although researchers have been studying stem cells from mouse embryos for more than 20 years, only recently have they been able to isolate stem cells from human embryos and grow them in a laboratory. In 1998, James A. Thomson of the University of Wisconsin, Madison, became the first scientist to do this. He is now at the forefront of stem cell research, searching for answers to the most basic questions about what makes these remarkable cells so versatile. Although scientists envision many possible future uses of stem cells for treating Parkinson’s disease, heart disease, and many other disorders affected by damaged or dying cells, Thomson predicts that the earliest fruits of stem cell research will be the development of powerful model systems for finding and testing new medicines, as well as for unlocking the deepest secrets of what keeps us healthy and makes us sick.


References edit

  1. U.S. Department of Health and Human Services. Inside the Cell. September 2005.<>.
  2. U.S. Department of Health and Human Services. Inside the Cell. September 2005.<>.

Cells Turn Nutrients into Usable Energy edit

Cells manage a wide range of functions in their tiny package — growing, moving, housekeeping, and so on — and most of those functions require energy. Cellular nutrients come in many forms, including sugars and fats. In order to provide a cell with energy, these molecules have to pass across the cell membrane, which functions as a barrier — but not an impassable one. Like the exterior walls of a house, the plasma membrane is semi-permeable. In much the same way that doors and windows allow necessities to enter the house, various proteins that span the cell membrane permit specific molecules into the cell, although they may require some energy input to accomplish this task.

Complex organic food molecules such as sugars, fats, and proteins are rich sources of energy for cells because much of the energy used to form these molecules is literally stored within the chemical bonds that hold them together. Scientists can measure the amount of energy stored in foods using a device called a bomb calorimeter. With this technique, food is placed inside the calorimeter and heated until it burns. The excess heat released by the reaction is directly proportional to the amount of energy contained in the food.

In reality, of course, cells don't work quite like calorimeters. Rather than burning all their energy in one large reaction, cells release the energy stored in their food molecules through a series of oxidation reactions. Oxidation describes a type of chemical reaction in which electrons are transferred from one molecule to another, changing the composition and energy content of both the donor and acceptor molecules. Food molecules act as electron donors. During each oxidation reaction involved in food breakdown, the product of the reaction has a lower energy content than the donor molecule that preceded it in the pathway. At the same time, electron acceptor molecules capture some of the energy lost from the food molecule during each oxidation reaction and store it for later use. Eventually, when the carbon atoms from a complex organic food molecule are fully oxidized at the end of the reaction chain, they are released as waste in the form of carbon dioxide.

Cells do not use the energy from oxidation reactions as soon as it is released. Instead, they convert it into small, energy-rich molecules such as ATP and nicotinamide adenine dinucleotide (NADH), which can be used throughout the cell to power metabolism and construct new cellular components. In addition, workhorse proteins called enzymes use this chemical energy to catalyze, or accelerate, chemical reactions within the cell that would otherwise proceed very slowly. Enzymes do not force a reaction to proceed if it wouldn't do so without the catalyst; rather, they simply lower the energy barrier required for the reaction to begin.

Metabolic Pathways edit

The living systems are highly ordered and utilize energy. This energy is not created by the living system. It is instead, obtain from the environment, and then processed into usable forms. Metabolism is a series of chemical reactions beginning with a particular molecule and converting it into another molecule or molecules. It has many defined pathways in the cells which are interdependent and their activity is coordinated very sensitively by means of communication in which allosteric enzymes are predominant.[1]

The overview of the process is that through photosynthesis, carbon dioxide and water, with the help of light, is converted into organic molecules, or food in our sense. Through cellular respiration, the organic molecules are converted back into carbon dioxide and water. Metabolism is the total chemical reaction occuring in the body, and it involves molecular interaction. It is highly regulated and yields a change in energy content of the reactants. The metabolic pathways is a series of reaction controlled by multiple enzymes.

Energy Transformation edit

Organisms transform energy, and energy is the capacity to do work. There are kinetic energies and potential energy. Kinetic energy is the energy of motion, it can be used to perform work. Examples of kinetic energy are light, heat, and electricity. Potential energy is stored energy. An example is electrochemical gradients. Light can be transformed into chemical bonds. Chemical bonds can be transformed to be used for mechanical work. Energy transformation must follow the two thermodynamic laws. The first law -conservation of energy - is that energy can neither be created or destroyed; it can only change forms. The Universe has a constant form of energy. The second law of that energy transformation yields an increase in the entropy of the universe. Entropy is a measure of disorder. But this is referring to the a closed system, one in which matter is isolated from the surrounding. So as long as the entropy of the system and surrounding increases, entropy of the system itself may decrease.

Classes edit

Catabolic Reactions edit

Catabolic Reactions are metabolic pathways in which reactions convert energy from fuels into biologically useful forms.

Anabolic Reactions edit

Anabolic Reactions are metabolic pathways in which reactions require energy to proceed.

Amphibolic Reactions edit

Amphibolic reactions are metabolic pathways that involves both catabolic and anabolic reactions.

Unifying Themes edit

Activated Carriers edit

ATP is an activated carrier of phosphoryl groups because phosphoryl transfer from ATP is an exergonic process. The use of activated carriers is a motif in biochemistry and most function as coenzymes:

1. Activated Carriers of Electrons for Fuel Oxidation. In aerobic organisms electrons are not transferred directly to O2 despite being the ultimate electron acceptor. Rather, fuel molecules transfer electrons to special carriers which are either pyridine nucleotides or flavens. The reduced form of these carriers then transfer their high-potential electrons to O2.

2. Activated Carrier of Electrons for Reductive Biosynthesis. High potential electrons are required in most biosyntheses because the precursors are more oxidized then the products. Hence, reducing power is needed in addition to ATP.

3. An Activated Carrier of Two-Carbon Fragments. Coenzyme A, another central molecule in metabolism is a carrier of acyl groups. Acyl groups are important constituents in both catabolism, and anabolism. The terminal sulfhydryl group of CoA is the reactive sites. Acyl groups are linked to CoA by thioester bonds resulting derivative called acyl CoA. The hydrolysis of thioester is thermodynamically favorable that that of an oxygen ester because the electrons of C=O cannot form resonance structures with C - bonds, making acetyl CoA have a high acetyl-group transfer potential because transfer of acyl group is exergonic. Acetyl CoA carries an activated acetyl group, just as ATP carries an activated phosphoryl group.


[13] Aceyl-CoA

Use of activated carriers illustrates that Kinetic stability of these molecules in the absence of specific catalysts is essential for their biological function because it enables enzymes to control the flow of free energy and reducing powers. Secondly most intercharges of activated groups in metabolisms are accomplished by rather small set of carriers such as ATP, NADH and NADPH. [2]

Vitamins edit

Almost all activated carriers that act as coenzymes are derived from vitamins. Vitamins are organic molecules that are needed in small amounts in the diets of some animals. They play the same roles in nearly all forms of life, but higher animals have lost the capacity to synthesize the in the course of evolutions.[3]


[14] Vitamin B6,coenzyme pyridoxal phosphate. Typical reaction type is group transfer to or from amino acids. Deficiency can lead to depression, confusion and convulsion.

Reiteration in Metabolism edit

Six fundamental reaction types that are the basis of metabolism:

1. Oxidation-reduction reactions are essential components of pathways. Useful energy is derived from the oxidation of carbon compounds.

2. Ligation reactions form bonds by using free energy from ATP cleavage

3. Isomerization reactions rearrange particular atoms within a molecule.

4. Group-transfer reactions play a variety of roles.

5. Hydrolytic reactions cleave bonds by the addition of water.

6. Functional groups may be added to double bonds to form single bonds or removed from single bonds to form double bonds.


Regulation in Metabolism edit

Metabolic reactions must be to rigorously related but at the same time must be flexible to adjust metabolic activity to constantly changing external environment cells. Metabolism are regulated through:

1. Controlling the Amounts of Enzyme. Amount of particular enzyme depends on both its rate of synthesis and its rate of degradation.

2. Controlling Catalytic Activity. Catalytic activity of enzymes are controlled in several ways:

    (a.) reversible allosteric control
    (b.) feedback inhbition
    (c.) reversible covalent modification
    (d.) hormones coordinate metabolic relations between different tissues
    (e.) energy status of cell --> energy charge 

3. Controlling the Accessibility of Substrate. Compartmentalization segregates opposed reactions and Controlling the flux of substrates.[5]

Metabolism from RNA World edit

The current thinking is that RNA was an early biomolecule that in an early RNA world would have served as a catalyst and information-storage. Activated carries such as ATP, NADH, FADH2 and coenzyme A contain adenosine diphosphate units evolved from early RNA catalyst. Non-RNA units such as isoalloxazine ring may have been recruited to serve as efficient carriers of activated electrons and chemical units, which were functions not performed by RNA itself. Then when a more versatile proteins replaced RNA as the major catalysts, the ribonucleotide coenzymes stayed essentially unchanged because they were already well suited to their metabolic roles. With the advent of protein enzymes, these important cofactors evolved as free molecules without losing adenosine diphosphate vestiage of their RNA-world ancestry explaining why molecules and motifs of metabolism are common to all forms of life.[6]

Thermodynamics of Pathways edit

Thermodynamically unfavorable reactions can be driven by a thermodynamically unfavorable reaction when it is coupled. This is because a pathway must satisfy two criteria: (1.) the individual reactions must be specific and (2.) the entire set of reactions that constitute the pathway must be thermodynamically favored. A reaction that is specific will yield only one particular product or set of products from its reactants due to enzyme specificity. Thermodynamics of metabolism is most readily approached in relation to free energy, which states that a reaction can occur spontaneously only if   is negative. This is due to the formation of products C and D from subtrates A and B given by the formula


Thus   depends on the nature of the reactants and their concentrations, which leads to thermodynamically fact that overall free-energy change for a chemically coupled series of reactions is equal to the free-energy changes of the individual steps.[7]

References edit

  1. Berg, Jeremy (2007). Biochemistry, 6th Edition. New York, New York: Sara Tenney. pp. 410–411. ISBN 978-0-7167-8724-2. {{cite book}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)
  2. Berg, Jeremy (2007). Biochemistry, 6th Edition. New York, New York: Sara Tenney. pp. 420–422. ISBN 978-0-7167-8724-2. {{cite book}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)
  3. Berg, Jeremy (2007). Biochemistry, 6th Edition. New York, New York: Sara Tenney. p. 423. ISBN 978-0-7167-8724-2. {{cite book}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)
  4. Berg, Jeremy (2007). Biochemistry, 6th Edition. New York, New York: Sara Tenney. pp. 425–427. ISBN 978-0-7167-8724-2. {{cite book}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)
  5. Berg, Jeremy (2007). Biochemistry, 6th Edition. New York, New York: Sara Tenney. pp. 428–429. ISBN 978-0-7167-8724-2. {{cite book}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)
  6. Berg, Jeremy (2007). Biochemistry, 6th Edition. New York, New York: Sara Tenney. p. 429. ISBN 978-0-7167-8724-2. {{cite book}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)
  7. Berg, Jeremy (2007). Biochemistry, 6th Edition. New York, New York: Sara Tenney. pp. 410–411. ISBN 978-0-7167-8724-2. {{cite book}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)

Adenosine triphosphate (ATP) is a nucleotide that consists of an adenine and a ribose linked to three sequential phosphoryl (PO32-) groups via a phosphoester bond and two phosphoanhydride bonds. ATP is the most abundant nucleotide in the cell and the primary cellular energy currency in all life forms. The primary biological importance of ATP rests in the large amount of free energy released during its hydrolysis. This provides energy for other cellular work, such as biosynthetic reactions, active transport, and cell movement. ATP is used in cellular metabolism in plants. It involved with light to create energy for plant. Besides, ATP is also one of components of DNA.

Definition edit

Chemical structure of ATP

ATP also known as adenosine 5'-triphosphate. It is formed from adenosine diphosphate (ADP) and orthosphosphate (Pi). When fuel molecules are oxidized in chemotrophs or when light is trapped by phototrophs. This nucleotide is tremendously important since it is the most commonly used energy currency. The energy is released from the cleave of the triphosphate group is used to power many cellular processes.[1]

Physical and Chemical Properties of ATP edit

ATP is composed of an adenine ring, ribose sugar, and three phosphate groups (triphosphate). The groups of the phosphate group are usually called the alpha (α), beta (β), and gamma (γ) phosphates. It is typically related to a monomer of RNA called adenosine nucleotide. Gamma phosphate group is the primary phosphate group on the ATP molecules that is hydrolyzed when the energy is needed to drive anabolic reactions. Basically gamma phosphate is typically located the farthest from the ribose sugar and has a higher energy of hydrolysis than either that of the alpha and beta phosphate. The bonds that are formed after hydrolysis or the phosphorylation of a residue by ATP are lower in energy than that of the phosphoanhydride bonds of ATP.

ATP is very soluble in water and is a quite stable solution that has a pH of 6.8-7.4, but is rapidly hydrolysed at extreme pH. Thus, ATP is best stored as an anhydrous salt.

Although, ATP is quite stable in solution, it is an unstable molecule in unbuffered water. This is because, once ATP gets in contact with unbuffered water, it hydrolyses to ADP and phosphate due to the strength of the bonds between the phosphate groups in ATP is commonly seen to be less than the strength of the hydrogen bonds (hydration bonds) between its products (ADP + phosphate) and water. Therefore, if ATP and ADP are in chemical equilibrium in water, almost all the ATP will form into ADP because of the reaction that will occur. Gibbs free energy is when a system is far from equilibrium and it is able to do some kind of work. It is seen that typical living cells maintain the ratio of ATP and ADP at a point ten orders of magnitude from equilibrium. However, this may only occur if ADP is thousand fold lower in concentration than that of ATP. This shows that hydrolysis of ATP in cells usually release a large amount of free energy in reaction.

However, even with releasing a large amount of free energy during reaction, any unstable system of potentially reactive molecules could potentially serve as a way of storing free energy. This is only if the cells maintain their concentration far from the equilibrium point of the reaction. However, the idea of both energy-release and entropy-increase always occur during the breakdown of RNA, DNA, and ATP into simpler monomers.

In an ATP molecule, two high-energy phosphate bonds called phsophoanhydride bonds are responsible for high energy content of this molecule. Based on biochemical reaction, these anhydride bonds are often referred to as high-energy bonds. Also the released of hydrolysis of the anhydride bonds can happen in the energy stored ATP.

Binding to Proteins edit

Rossmann fold is a type of protein fold that some proteins and ATP bind together as. This characteristic protein fold is a general nucleotide-binding structural domain that can also bind the coenzyme NAD. Kinase is the most common ATP-binding protein. They share a small number of common folds and there biggest kinase superfamily all share common structural features specialized for ATP binding and phosphate transfer.

ATP also requires the presence of a divalent cation that is almost as magnesium as a metal used. This metal binds to the ATP phosphate groups. This metal ion can also serve as a mechanism for kinase regulation. The presence of magnesium greatly decreases the dissociation constant of ATP from its protein binding partner without even affecting the ability of the enzyme to catalyze its reaction once the ATP has bound.

Intracellular ATP edit

Intracellular ATP hydrolysis is catalyzed by intracellular ATPases. For example, the (Na+-K+)-ATPase located in the plasma membranes of higher eukaryotes drives active transport of Na+ and K+ coupled to ATP hydrolysis, and generates electrochemical gradients across the cell membrane. Another important intracellular ATPase is myosin. The myosin heads form the cross-bridges to thin filaments in intact myofibrils and its ATP-powered movement is responsible for muscle contraction.

Extracellular ATP edit

ATP is also present in extracellular spaces in nanomolar to micromolar concentrations, which are 3-6 orders of magnitude lower than intracellular ATP concentration (1, 2). ATP is released from cells to extracellular spaces by regulated exocytosis or plasma membrane channels (Figure 1). Regulated exocytosis is an important process used to release substances such as hormones or neurotransmitters from the cell and is triggered by an increase in cytoplasmic Ca2+ concentration (2, 3). ATP efflux also occurs through plasma membrane conductance channels, transporters, or constitutive secretory pathways as residual cargo products (2). Extracellular ATP acts as a neurotransmitter and an autocrine/paracrine chemical messenger in non-neural tissues. Its effects are mediated by the P2 purinergic receptors and elicit a variety of physiological responses, such as neurotransmission, regulation of secretion, modulation of immune functions, pain transmission, apoptosis etc.

P2 receptors consist of two major subfamilies, P2X and P2Y. P2X receptors are ligand-gated ion channels and P2Y receptors are G protein-coupled receptors. The concentration of extracellular ATP is regulated by its hydrolysis that is catalyzed by extracellular ATPases. Thus the physiological responses mediated by the purinergic receptors are modulated by extracellular ATPases (Figure 2). For example, Sesti et al. reported that ATP modulates norepinephrine release from cardiac sympathetic nerve endings and this action of ATP is controlled by purinergic receptors in cardiac synaptosomes and modulated by extracellular ATPases (4). Di Virgilio et al. reported that a potent platelet aggregating factor is ADP and its amount is regulated by the activity of extracellular ATPases on endothelial cells (5). However, the precise relationship of multiple P2X and P2Y receptor subtypes to extracellular ATPases remains to be determined.

Exergonic Reaction edit

The role of ATP is an energy-rich molecule because its triphosphate unit contains two phosphoanhydride bonds. Large amounts of free energy is liberated when ATP is hydrolyzed to adenosine diphosphate (ADP) and orthosphosphate (Pi) or when ATP is hydrolyzed to adenosine monophosphate (AMP) and pyrophosphate (PPi). The precise   for these reactions depend on ionic strength of the metal such as Mg 2+. The free energy is liberated in hydrolysis of ATP is harnessed to drive reactions that require an input of energy for muscle contraction. The formation of ATP from ADP and Pi is known as the ATP-ADP cycle is the fundamental mode of energy exchange in biological systems. It is intriguing to note that although, all nucleotide triphosphates are energetically equivalent, ATP is the primary cellular energy carrier. Under cellular conditions, the hydrolysis of ATP shifts the equilibrium of a coupled reaction by a factor of 108[2]

Phosphoryl Potential edit

ATP has a particularly efficient phosphoryl-group donor that can best be explained by features of the ATP structure:

Resonance Structures. ADP and Pi have greater resonance stabilization than does ATP. Orthophosphate has multiple resonance forms of similar energy whereas the   phosphoryl group of ATP has a smaller number due to its unfavorability of the positively charged oxygen atom that is adjacent to a positively charged phosphorus atom.

Electrostatic Repulsion. At pH 7, triphosphate unit of ATP carries four negative charges which repel one another due to their close proximity. The repulsion between them is reduced when ATP is hydrolyzed.

Stabilization Due to Hydration. More water can bind effectively to ADP and Pi than can bind to the phosphoanhydride part of ATP, stabilizing the ADP and Pi by hydration. [3]

Consumption of ATP edit

The large amounts of energy provided by the hydrolysis of ATP are necessary to overcome the large free energy changes necessary to create the large macromolecular proteins. The cleavagle of the phosphoanhydride bonds in ATP provides the source for free energy to make biological reactions spontaneous (negative free energy). Because the amount of entropy of the universe is continually increasing it is unfavorable for large macromolecules to form without the use of ATP. Because of this, the free energy generated by the ATP is always immediately consumed by nearby endergonic (energy-reguiring) biological reactions. The exergonic reaction of the ATP is only able to proceed if it is coupled to an endergonic reaction, otherwise thermodynamic equilibrium would not be obtained. The consumption of ATP proceeds with the first step of having an enzyme attache an amino acid to the a-phosphate of ATP. This results in the release of a pyrophosphate. This release is called an aminoacyl-adenylate intermediate. The reaction then proceeds to the enzyme catalyzing transfer of an amino acid to one of two -OH locations on the ribose portion of the adenosine residue. ATP is able to release energy into cells because cells maintain a concentration of ATP that is far higher above the equilibrium concentrations. The high concentration of ATP allows it to be the main provider of driving endergonic reactions in cells. This coupling of energy releasing and consuming systems through a common intermediate is vital to energy exchange in living systems. [4] = Lehninger | firs = Albert | authorlink = |Nelson, David L. and Michael M. Cox | title = Lehninger principles of biochemistry, 4th ed | publisher = W.H. Freeman & Co | date = 2007 | location = New York, New York | pages 22–25 | isbn = 0-7167-4339-6 }}</ref>

Importance of Oxidation of Carbon edit

Formation of ATP edit

ATP is a principal immediate donor of free energy in biological systems meaning that it is consumed within a minute of it formation. The carbon in fuel molecules such as glucose and fats are oxidized to CO2 and the energy released is used to regenerate ATP from ADP and Pi. Oxidation in fuel takes place one carbon at a time and the carbon-oxidation energy is used in some cases to create compounds with high phosphoryl-transfer potential and other cases to create ion gradient as well with the end formation of ATP.[5]

Coupling with Carbon Fuels edit

ATP is coupled with oxidation of carbon fuels directly and through the formation of ion gradients. Energy of oxidation is initially trapped as high-phosphoryl-transfer potential compound and then used to form ATP. In ion gradients the electrochemical potential, produced by oxidation of fuel molecules or by photosynthesis, which ultimately powers the synthesis of most ATP in cells. ATP hydrolysis can be used to form ion gradients of different types and functions.

Energy from Food edit

Described by Hans Krebs the three stages in generation of energy from oxidation of foodstuffs:

1. Large molecules in foods are broken down into smaller units in a process known as digestion. Proteins are hydrolyzed to their 20 different amino acids, polysaccharides are hydrolyzed into simple sugars and lastly fats are hydrolyzed to glycerol and fatty acids.

2. Numerous small molecules are degraded to a few simple units that play a central role in metabolism. Sugars, fatty acids, glycerol and several amino acids are converted into the acetyl unit of acetyl CoA. Some ATP is generated but not a substantial amount.

3. ATP is produced from the complete oxidation of acetyl unit of acetyl CoA. Final stage consist of citric acid cycle and oxidative phosphorylation which are the final pathways in oxidation of fuel molecules. Acetyl CoA brings acetyl units into the citric acid, where they are completely oxidized to CO2. Four pairs of electrons are transferred for each acetyl group that is oxidized. Then a proton gradient is generated as electron flows from the reduced forms of these carriers to O2 and the gradient is used to synthesize ATP.[6]



References edit

  1. Berg, Jeremy (2007). Biochemistry, 6th Edition. New York, New York: Sara Tenney. pp. 110–111. ISBN 978-0-7167-8724-2. {{cite book}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)
  2. Berg, Jeremy (2007). Biochemistry, 6th Edition. New York, New York: Sara Tenney. pp. 413–415. ISBN 978-0-7167-8724-2. {{cite book}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)
  3. Berg, Jeremy (2007). Biochemistry, 6th Edition. New York, New York: Sara Tenney. p. 415. ISBN 978-0-7167-8724-2. {{cite book}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)
  4. Biochemistry, 6th Edition. New York, New York: Sara Tenney. 2007. p. 110. ISBN 978-0-7167-8724-2. {{cite book}}: |first= missing |last= (help); Unknown parameter |= ignored (help); Unknown parameter |coauthors= ignored (|author= suggested) (help)
  5. Berg, Jeremy (2007). Biochemistry, 6th Edition. New York, New York: Sara Tenney. p. 417. ISBN 978-0-7167-8724-2. {{cite book}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)
  6. Berg, Jeremy (2007). Biochemistry, 6th Edition. New York, New York: Sara Tenney. pp. 419–420. ISBN 978-0-7167-8724-2. {{cite book}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)

1. Schwiebert, E. M. ABC transporter-facilitated ATP conductive transport. Am. J. Physiolo., 1999, 276, C1-C8.

2. Lazarowski, E. R., Boucher, R. C., and Harden, K. T. Mechanisms of release of nucleotides and integration of their action as P2X- and P2Y-receptor activating molecules. Mol. Pharmacol., 2003, 64, 785-795.

3. Theander, S., Lew, D. P., and Nüße, O. Granule-specific ATP requirements for Ca2+-induced exocytosis in human neutrophils. Evidence for substantial ATP-independent release. J. Cell Sci., 2002, 115, 2975-2983.

4. Sesti, C., Broekman, M. J., Drosopoulos, J. H., Islam, N., Marcus, A. J., and Levi, R. Ectonucleotidase in cardiac sympathetic nerve endings modulates ATP-mediated feedback of norepinephrine release. J. Pharmacol. and Exp. Ther., 2002, 300, 605-611.


6. Di Virgilio, F., Chiozzi, P., Ferrari, D., Falzoni, S., Sanz, J. M., Morelli, A., Torboli, M., Bolognesi, G., and Baricordi, O. R. Nucleotide receptors: an emerging family of regulatory molecules in blood cells. Blood, 2001, 97, 587-600.

Acetyl-CoA edit

Acetyl coenzyme A, or better known as acetyl-CoA, is an important molecule used in metabolic processes. It is primarily used by the body for energy production through the citric acid cycle, or Krebs cycle.

Formation edit

Acetyl-CoA is a product of the oxidation of several amino acids, pyruvate and fatty acids. It is formed after pyruvate enters the mitochondria via active transport. The oxidation transformation that converts pyruvate into acetyl-CoA is known as a pyruvate dehydrogenase reaction. In this process, each pyruvic acid molecule loses one carbon atom, and combines with oxygen to produce carbon dioxide. This carbon dioxide is then exhaled out of the cell. This process is catalyzed by a complex of three enzymes through a process known as pyruvate dehydrogenase complex (PDC). [1]

Functions edit

The main function of Acetyl-CoA is to carry acyl groups or thioesters. It is the precursor to HMG CoA, an important part of cholesterol and ketone synthesis. It can also be found as a vital reagent in the synthesis of fatty acids and sterols, as well as the oxidation of fatty acids as well as the breaking down of many amino acids. [2]

Acetyl-CoA is well known as the junction between Glycolysis and the Citric Acid Cycle as well as an essential component in balancing between carbohydrate and fat metabolism. Acetyl-CoA has also been a central metabolite that is involved in many metabolic transformations within the cell. The acetyl group of the acetyl-CoA is used to oxidize via the TCA cycle to reduce NAD+ and FAD to NADH and FADH2, respectively. These products are then used to fuel ATP production through the electron transport train.

In May 2011, Ling Cai et al. found that Acetyl-Coa functioned as a carbon-source rheostat that signals the initiation of the cellular growth program by promoting the acetylation of histones specifically at growth genes. By intimately coordinating with nutrient availability, the acetyl-CoA was concluded to play as a critical signal for metabolic growth and proliferation.

Activation of acetyl-CoA: Some acetyl groups within the cell are tagged with coenzyme A (CoA). Acetyl-CoA molecules are tagged at the back end by a process called condensation with carbon dioxide, catalyzed by acetyl-CoA carboxylase in order to redirect acetyl groups into fatty acid biosynthesis.

References edit


Glycolysis and Gluconeogenesis edit

Glycolysis/Gluconeogenesis is the set of reactions that converts glucose into pyruvate. Glycolysis is a compilation of ten reactions (with 1 being an isomerization of 2 products into one another) of glycolysis take place in the cytoplasm.

  1. One glucose molecule will go under group-transfer reaction with 1 ATP (Adenosine Triphosphate) - which is catalyzed by hexokinase - to become Glucose 6-phosphate (Glu 6-P) and produce 1 ADP (adenosine diphosphate) molecule.
  2. Glu 6-P, under the activity of the enzyme phosphoglucose isomerase, becomes another isomer, Fructose 6-Phosphate (Fru 6-P).
  3. Then, the group-transfer reaction between Fru 6-P and ATP, catalyzed by the enzyme Phosphofructokinase-1 (known as PFK-1), produces 1 fructose 1,6-biphosphate (Fru 1,6-P) molecule and another ADP molecule.
  4. Fruc 1,6-P (after ring opening), after binding to the enzyme aldolase, produces two new molecules known as:
    1. Glyceraldehyde 3-phosphate (GAP)
    2. Dihydroxyacetone phosphate (DHAP)
    3. Note that GAP can isomerize to DHAP and vice versa in the reaction catalyzed by the enzyme Triose Phosphate Isomerase.
  5. GAP, which is constantly supplemented by the isomerization of DHAP, continues the process of glycolysis by reacting with 2 inorganic phosphorus molecules and 2 nicotinamide adenine dinucleotide (NAD+, oxidized form) molecules, under oxidation-reduction reactions catalyzed by the enzyme Glyceraldehyde Phosphate Dehydrogenase, to produce 2 1,3-bisphosphate glycerate molecules, 2 protons, and 2 NADH molecules (reduced form of NAD+). Note that from this stage on, reactions are carried out by 2 molecules of the previous products to produce 2 new molecules. Therefore, it is assumed in the next stages, "1,3-biphosphate glycerate reacts with some compound A" means 2 1,3-bisphosphate glycerate molecules react with 2 molecules of compound A to produce 2 new molecules.
  6. Next, 1,3-bisphosphate glycerate undergoes group-transfer reaction, catalyzed by phosphoglycerate kinase, with ADP to produce 3-phosphoglycerate and ATP.
  7. Then, 3-phosphoglycerate, catalyzed by phosphoglycerate mutase, becomes 2-phosphoglycerate.
  8. Catalyzed by the enzyme enolase, 2-phosphoglycerate produces phosphoenolpyruvate (PEP) and water.
  9. In the last step, PEP reacts with ADP in a group-transfer reaction catalyzed by phosphopyruvate kinase, to produce pyruvate and ATP.

In total, glycolysis consumes 1 glucose molecule, 2 ATP molecules, and 2 NAD+ to produce 2 pyruvate molecules, 4 ATP molecules, and 2 NADH molecules.

Therefore, in overall, 1 Glu + 2 NAD+2 Pyruvate + 2 NADH + 2 ATP

After glycolysis, the products, depending on the appearance of O2, will undergo either aerobic reaction (with O2) to continue the metabolic pathway into the citric acid cycle (also known as Kreb's cycle), or anaerobic reaction (without O2) to start a new process known as fermentation to produce lactic acid (mostly in human's muscular cells) or ethanol and carbon dioxide (CO2) in microbes such as yeasts.

Disaccharide Metabolism edit

Lactose, sucrose, and maltose are disaccharides that can be hydrolyzed into monosaccharaides and then enter the glycolysis. Latcase, sse, and maltase are enzymes(in epithelial cells) that can hydrolyze the disaccharides respectively. Maltose is hydrolyzed into two β-D-glucopyranose molecules. These monosaccharaides can readily enter glycolytic pathway.

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Hydrolyzing sucrose produces α-D-glucopyranose and β-D-fructofuranose. However, β-D-fructofuranose must be converted to a form in order to enter glycolytic. This conversion is facilitated by the enzyme hexokinase to form β-D-fructofuranose-6-phosphate or by the fructokinase to form β-D-fructofuranose-1-phosphate. Both of these phosphorylation reactions consume ATP.

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Constituent monosaccharaides residues of lactose are β-D-glucopyranose and β-D-galactopyranose. β-D-glucopyranose is ready to enter into glycolytic process but β-D-galactopyranose needs to convert to a form to enter glycolytic pathway. Since galactose is a epimer of glucose at the C-4 position, it can form glucose-6-phosphate (an intermediate reactant) during glycolysis. [1]

Glycolysis as Survival Pathway for Brain Cells edit

The brain uses a very large amount of energy, and neurons and astrocytes are mainly responsible for the consumption of oxygen and glucose. The brain is only ~2% of the human body weight, though it consumes over 20% of the body’s oxygen and glucose.

When nitric oxide inhibits respiration in neurons of rats, ATP concentration quickly decreases along with a sharp decrease in mitochondrial transmembrane potential, followed by spontaneous apoptosis or cell death. Astrocytes respond to respiration inhibition by instead increasing metabolism through glycolysis and using the generated ATP to maintain the mitochondrial transmembrane potential. This is done through an increase in the enzyme activity of PFK1 or 6-phosphofructo-1-kinase, independently of cGMP, at a level twice that of normal neuron conditions. Levels of fructose-2,6-bisphosphate rise in astrocytes as well, which also activates PFK1 allosterically. However, levels of this enzyme are NOT raised in the neuron cells. The maintenance of the potential prevents cell apoptosis.

The mechanism that increases the levels of these enzymes in astrocrytes during conditions of respiration inhibition is quick phosphorylation of the enzyme AMPK, or 5’-AMP-activated protein kinase and the gathering of fructose-2,6-bisphosphate. This shows that when respiration is inhibited a ‘crisis’ has occurred causing a cascade of events: 5’-AMP concentration increase, causing phophorylation of AMPK, which activates fructose-2,6-bisphosphatase.

Neurons however are unable to block cell death through an increase in glycolytic activity due to this lack of 6-phosphofructo-2-kinase and fructose-2,6-bisphotase. Instead the metabolism of glucose is achieved via the pentose phosphate pathway, which regenerates reduced glutathione. [2]

References edit

1. Campbell, Neil A. (2005). Biology. Pearson. ISBN 0-8053-7146-0. {{cite book}}: Check |isbn= value: checksum (help); Text "coauthors+ H.C. Van Ness, M.M. Abbott" ignored (help)

2. Bolaños, J. SY. “Glycolysis: a bioenergetic or a survival pathway?” Trends in Biochemical Sciences, Volume 35, Issue 3, 145-149, 18 December 2009

Definition edit

Example of Anaerobic Respiration

Anaerobic respiration is the formation of ATP without the presence of oxygen. This method uses the electron transport chain without the presence of oxygen as the electron acceptor. Although oxygen is highly oxidizing, it is only used during aerobic processes. In anaerobic repiration, less oxidizing molecules such as sulfate (SO42-), nitrate (NO3-), or sulfur (S) are used as electron acceptors. Thus, less energy is formed per molecule of glucose during anaerobic respiration. Most prokaryotes that live under environmental conditions that lack oxygen uses aerobic respiration, although humans too, use it sometimes as well (Lactic Acid Fermentation).

In this process, specifically with the absence of Oxygen during respiration process, organisms have evolved with mechanisms to recycle Nicotinamide Adenine Dinucleotide (NAD+) for glycolysis to continue in order to synthesize Adenosine Triphosphate (ATP) molecules, known as "energy currency" of cells. This process evolves into two different mechanisms, although both share the name of "fermentation":

  1. Ethanol Fermentation occurs in bacteria, yeast,...
  2. Lactic Acid (or Lactate) Fermentation occurs in animals (humans,...)

Ethanol Fermentation edit

After glycolysis, without Oxygen, pyruvates are carried into the ethanol fermentation cycle:

  1. With cofactors such as pyruvate decarboxylase enzyme, TPP (thiamine pyrophosphate), pyruvate is converted into acetaldehyde and carbon dioxide.
  2. Acetaldehyde, under oxidation-reduction reaction catalyzed by the enzyme ethanol dehydrogenase, is reduced to ethanol, with NADH being oxidized to NAD+.
  3. NAD+ is then used for glycolysis to synthesize ATP, and if Oxygen is still absent, fermentation continues. Therefore, ethanol and CO2 are waste products of this fermentation process.

Note that because of this reaction, when using yeast or some bacteria in food processing, there are a slight smell of alcohol (from ethanol) and gas coming off the mixture (from CO2)

Lactic Acid Fermentation edit

Example of Lactic Acid Fermentation

Similarly, in animals (humans,..), pyruvates must go into fermentation cycle if no oxygen is carried into the cells. However, there is one difference:

Under reduction-oxidation reaction catalyzed by the enzyme lactate dehydrogenase, pyruvate is reduced into lactate, with NADH being oxidized to NAD+.

Note that in animals, overworked muscles, whose cells cannot receive sufficient amount of oxygen to compensate for the loss of ATP, produce a lot of lactic acid as waste product of this fermentation process. Fermentation is diverse. There are two types of fermentation: homofermentative and heterofermentative. Homofermentative include lactic acid, ethanol, and carbon dioxide. However, heterofermentative include propionic acid, acetic acid, carbon dioxide, hydrogen gas, butyric acid, butanol, acetone, isopropyl alcohol, and succinic acid. Furthermore, in lactic fermentation there are homolactic versus heterolactic. The heterofermentative lactic acid bacteria lack aldolase and that the first steps of heterofermentative pathway are from the Pentose-Phosphate Pathway. An example of lactic fermentation is an organism called Lactobacillus acidophilus. This organism is a gram positive, it is homofermentative, it is belong to Firmicutes group, and it can be found in the normal human flora.

Fats and Protein edit

Sometimes, glucose may not be available for cellular respiration. Without this essential fuel, cellular respiration would stop completely and death would occur. So there must be backup storage that keep fuel just in case glucose levels are insufficient. When glucose is low, the body uses carbohydrates, such as glycogen stored in the liver and muscles. When carbohydrates deplete, fat is used next and, as last resort, protein is used. When fats and proteins are used, they must first be converted to glucose or some derivative. Fats are broken down to fatty acids and glycerol. Fatty acids can turn into acetyl-CoA by beta oxidation. Glycerol can turn into an intermediate of glycolysis. Proteins are broken down into amino acids. They are modified into -keto acids then converted into the various intermediates of Kreb's cycle.

References edit


Microbiology. Spencer (TA). Microbiology 120 Lecture. 11/6/12

Introduction edit

In the aerobic respiration is a release of energy from glucose or another organic substate in the presence of Oxygen. Also, aerobic respiration is in the absent of air. Aerobic respiration is a process of cellular respiration that uses oxygen in order to break down molecules, which then release the electrons and also create the energy. It creates substances known as ATP. ATP stands for adenosine triphosphate. ATP's role is to store and carry most of the energy to other body cells. Besides, there are two main byproducts of aerobic respiration. They are water and carbon dioxide. Aerobic respiration is also contains three stages: glycolysis, Kreb's cycle, and the third stage is the electron transport phosphorylation.

The pyruvate produced in glycolysis undergoes further breakdown through a process called aerobic respiration in most organisms. This process need oxygen and yields much more energy than glycolysis. Aerobic respiration is separated into two processes: the Krebs cycle, and the Electron Transport Chain, which produces ATP through chemiosmotic phosphorylation. The energy conversion is as follows:

C6H12O6 + 6O -> 6CO2 + 6H2O + energy (38ATP)

Krebs Cycle edit

The Krebs cycle occurs in the mitochondria of a cell. Before entering the Krebs cycle, the pyruvic acid molecules are altered. During the process, the pyruvic acid molecule is broken down by an enzyme, one carbon atom is released in the form of carbon dioxide, and the remaining two carbon atoms are combined with a coenzyme called the coenzymes. After the glycolysis takes place in the cell's cytoplasm, the pyruvic acid molecules travel into the interior of the mitochondrion. Once the pyruvic acid is inside, carbon dioxide is enzymatically detached from each three-carbon pyruvic acid molecule to form acetic acid. The enzyme then syndicates the acetic acid with coenzyme A to produce acetyl CoA. Once acetyl CoA is formed, the Krebs cycle begins. The cycle is split into eight steps, picture of this cycle is included in this link:

Prior to entering the Krebs Cycle, pyruvate must be converted into acetyl CoA (pronounced: acetyl coenzyme A). This is achieved by removing a CO2 molecule from pyruvate and then removing an electron to reduce an NAD+ into NADH. An enzyme called coenzyme A is combined with the remaining acetyl to make acetyl CoA which is then fed into the Krebs Cycle. 1. Citrate is created when the acetyl group from acetyl CoA combines with oxaloacetate from the previous Krebs cycle. 2. The citric acid molecule experience an isomerization. A hydroxyl group and a hydrogen molecule are detached from the citrate structure in the form of water. The two carbons form a double bond until the water molecule is added again. Only now, the hydroxyl group and hydrogen molecule are reversed with respect to the original structure of the citrate molecule. Thus, isocitrate is formed. 3. Isocitrate is oxidized to form the 5-carbon α-ketoglutarate. This step releases one molecule of CO2 and reduces NAD+ to NADH2+. 4. The α-ketoglutarate is oxidized to succinyl CoA, yielding CO2 and NADH2+. 5. Succinyl CoA releases coenzyme A and phosphorylates ADP into ATP. 6. Succinate is oxidized to fumarate, converting FAD to FADH2. 7. An enzyme adds water to the fumarate molecule to form malate. The malate is formed by adding one hydrogen atom to a carbon atom and then adding a hydroxyl group to a carbon next to a terminal carbonyl group. 8. Malate is oxidized to oxaloacetate, reducing NAD+ to NADH2+.

Unlike anaerobic respiration, in this process, where oxygen appears in sufficient amount, pyruvates are transported into mitochondria, where the largest ATP-producing facilities will provide energy for the cell. Aerobic Respiration consists of three main steps:

  1. Pyruvate dehydrogenase complex
  2. Krebs Cycle (Nitric Acid Cycle)
  3. Oxidative Phosphorylation Cycle (Electron Transport Chain cycle)

Reference edit

Overview edit

This process involves with the conversion of pyruvate molecule into compound called acetyl coenzyme A, or acetyle CoA. This step is the junction between glycolysis and the Krebs Cycle (Citric Acid cycle) and is accomplished by a multi-enzyme complex that catalyzes three reactions:


  1. Pyruvate's carboxyl group (COO), which is fully oxidized and is removed to release CO2
  2. The remainning two-carbon is oxidized and form a compound named acetate. An enzyme transfers the extracted electrons to NAD+, storing energy in the form of NADH
  3. Finally, coenzyme A(CoA), a sulfur-containing compound derived from a B vitamin, is attached to the acetate by an unstable bond and this makes the acetyl group become very reactive. acetyl CoA has a high potential energy will undergoes the Citric Acid cycle to release energy to make ATP.

Reference edit

1. Reece, Jane B., and Neil A. Campbell. Campbell biology Jane B. Reece ... [et al.].. 9th ed. San Francisco: Benjamin Cummings :, 2011. Print.

Citric acid cycle

The Citric Acid Cycle has eight-steps.

Citric Acid Cycle edit

Other name for citric acid cycle is tricarboxylic acid (TCA) cycle or the Krebs cycle. The citric acid cycle is the central metabolic core of the cell. It is the final common pathway for oxidation — in other words harvesting high energy electrons--fuel molecules such as carbohydrate fatty acids, and amino acids by entering the cycle as Acetyl Coenzyme A (CoA). This reaction takes place inside of mitochondria. It is very efficient because it can generate large amounts of NADH and FADH. The citric acid cycle provides the majority, 90 percent, of energy used by aerobic human cells. By acting as the first stage of cellular respiration, the generation of high energy electrons from the citric acid cycle, in turn, are used in oxidative phosphorylation to reduce O2, generate proton gradient, and, later, the synthesis of ATP.

Citric Acid Cycle Links to Glycolysis by Pyruvate Dehydrogenase edit

Carbohydrates are mostly processed by glycolysis into pyruvate. Depending on the organism, the pyruvate is converted into either lactate or ethanol under anaerobic conditions. Through a specific carrier protein embedded in the mitochondrial membrane, pyruvate is transported into the mitochondria under aerobic conditions. Then, the pyruvate is oxidatively decarboxylated by the pyruvate dehydrogenase complex in order to form the acetyl CoA in the mitochondrial matrix. The reaction is shown in the following:
Pyruvate + CoA + NAD+ → acetyl CoA + CO2 + NADH + H+
This is an irreversible reaction which links glycolysis and the citric acid cycle together. As shown, CO2 is produced by pyruvate dehydrogenase complex and it captures high-transfer-potential electrons in the form of NADH. Therefore, the pyruvate dehydrogenase relates to the reactions of the citric acid cycle itself. The pyruvate dehydrogenase complex is composed of three different enzymes. Its complex is composed of members of a family of homologous complexes which include citric acid cycle enzyme a-ketoglutarate dehydrogenase complex. These complexes are very large, even bigger than ribosomes, with its molecular mass to be in between 4 million to 10 million daltons.

Reactions edit

Acetyl-CoA, main product of the Pyruvate Dehydrogenase Complex in aerobic respiration, starts the Krebs cycle. (An irreversible reaction that is link between glycolysis and the citric acid cycle.) The mechanism of the synthesis of acetyl coenzyme A from pyruvate requires five coenzymes and three enzymes. It is a very complex mechanism which many enzymes and coenzymes. Cofactors, which function as substrates, are divided into two different cofactors which are catalytic cofactor and stoichiometric cofactor. The catalytic cofactor includes coenzymes such as thiamine pyrophosphate (TPP), lipoic acid, and FAD. The stoichiometric cofactor includes coenzymes such as CoA and NAD+. Pyruvate converts into acetyl CoA in three distinct steps which include: decarboxylation, oxidation, and transfer of the resultant acetyl group to CoA.

Pyruvate results in formation of acetyl-CoA by a three step reaction:

1. Decarboxylation: TPP is combined with the pyruvate and decarboxylated in order to yield hydroxyethyl-TPP. Of the pyruvate dehydrogenase component, TPP is known as the prosthetic group which the carbon atom between the nitrogen and sulfur atoms in the thizaole ring is more acidic than most double bonded carbon groups with pKa values near 10. This reaction is catalyzed by the (E1) pyruvate dehydrogenase component of the multienzyme complex. The carbon center located in the TPP is ionized to form a carbanion which is added to the carbonyl group of pyruvate. As part of decarboxylation, a positive charged ring of TPP stabilizes the negative charge which was transferred to the ring. Finally, the protonation yields hydroxyethyl-TPP.

  Pyruvate +  TPP( conenzyme thiamine pyrophosphate)+ 2 H+ --> Hydroxyehthyl-TPP + CO2

2. Oxidation: In order to form an acetyl group, the hydroxyethyl group which is attached to TTP is oxidized. Simultaneously, the hydroxyethyl group is transferred to lipoamide which is lipoic acid derived that links to the side chain of a lysine residue by an amide linkage. This creates the formation of an energy-rich thioester bond. In this reaction, the disulfide group of lipoamide acts as an oxidant and is reduced to the disulfhydryl form. This reaction is catalyzed by the pyruvate dehydrogenase component (E1) as well and yields the acetyllipoamide.

  Hydroxyethyl-TPP + Lipoamide --> TPP + Acetyllipoamide

3. Formation of Acetyl CoA: Formation of Acetyl CoA: In this step, acetyl CoA is formed when acetyl group is transferred from acetyllipoamide. This reaction is catalyzed by dihydrolipoyl transacetylase (E2). As the acetyl group is transferred to the CoA, the energy-rich thioester bond is preserved. Thus, the fuel for the citric acid cycle, acetyl CoA has been generated from pyruvate for use. Until the dihydrolipoamide is oxidized to lipoamide, the pyruvate dehydrogenase complex cannot complete another catalytic cycle.

  CoA + Acetyllipoamide --> Acetyl CoA + Dihydrolipoamide

4. Formation of NADH: The final step in this reaction occurs when the oxidized form of lipoamide is regenerated by dihydrolipoyl dehydrogenase (E3).Two electrons are transferred to first an FAD prosthetic group of the enzyme and then to NAD+. This process of transferring electron is very unusual because FAD are known to receive electrons from NADH, not transfer them. Within the enzyme, the electron-transfer potential of FAD is increased by its chemical environment which enables it to transfer electrons to NAD+. Flavoproteins are proteins which are tightly associated with FAD or FMN also known as flavin mononucleotide.

  Dihydrolipoaminde + FAD --> Lipoamide + FADH2 + NAD+ --> FAD + NADH + H+

Overall reaction:

  Pyruvate + CoA + NAD+ --> acetyl CoA + CO2 + NADH +H+

Citric Cycle Reactions edit

1. The first reaction of the cycle is condensation of acetyl-CoA with oxaloacetate to form citrate. In this reaction, acetyl group is joined to the carbonyl group of oxaloacetate. The reaction between acetyl-CoA with oxaloacetate is necessary to form an active site closed citryl CoA complex for hydrolysis because the active site of acetyl-CoA with hydrolysis is a wasteful process.

  Oxaloacetate + Acteyl-CoA --> Citryl-CoA + H2O -->Citrate + CoA

2. Formation of Isocitrate via cis-Aconitate: Enzyme, aconitase catalyzes the reversible transformation of citrate to isocitrate. This is actually a 2 steps mechanism, which interchange an hydrogen with an hydroxyl group. First citrate is dehydrated to form the intermediary formation of the tricarboxylic acid cis-aconitate. Then by using the same enzyme, aconitase, isocitrate is formed. Aconitase is an iron-sulfur protein that participate in the dehydration and rehydration of the substrate.

  Citrate <-(forward rxn removes water)-> cis-Aconitate <-(forward react with water)-> Isocitrate

3. Oxidation of ioscitrate with NAD+ to α-Ketoglutarate, CO2 and NADH. Isocitrate dehydrogenase catalyzed oxidative decarboxylation of isocitrate to form α-ketoglutarate. Mg2+ is used to interact with the carbonyl group of oxalosuccinate. The rate of formation of α-ketoglutarate determines of overall reaction in the citric cycle. The intermediate, oxalosuccinate, is a very unstable β-ketoacid. Reaction with Intermediate:

  Isocitatrate + NAD+ --> Oxalosuccinate + NADH + H+
  Oxalosuccinate + H+ --> CO2 + α-ketoglutarate

Overall Reaction:

  Isocitrate + NAD+ --> α-ketoglutarate +CO2 + NADH

4. Oxidation of α-ketoglutarate to succinyl-CoA and CO2. This is another oxidative decarboxylation. Alpha-ketoglutarate is converted to succinyl-CoA and CO2 by the action of the α-ketoglutarate dehydrogenase complex. NAD+ is used as electron acceptor and CoA as the carrier of the succinyl group. This reaction is virtually identical to the pyruvate dehydrogenase reaction. It has three enzymes participating in this step. This is similar to the Pyruvate Dehdrogenase Complex reaction.

  α-ketoglutarate+ CoA + NAD+ -(α-ketoglutarate dehydrogenase complex)-> succinyl-CoA + CO2A +NADH

5. Conversion of Succinyl-CoA to Succinate: Succinyl-CoA has a thioester bond. Thioester bond retains a lot of energy (ΔG° = -33.5 kJ mol -1. Energy is released by breaking the thioester bond. The enzyme that is used in this step is called succinyl-Co synthetase or succinic thiokinase. The cleavage of the bond is coupled with GDP getting phosphorylated.

  Succinyl CoA + Pi + GDP --> succinate + CoA + GTP

6. Oxidation of succinate to fumarate: the succinate formed from succinyl-CoA is oxidized to fumarate by the enzyme called succinate dehydrogenase. FAD, which is attached to histidine side chain, acts as the electron acceptor by removing two hydrogen from succinate. FADH2 will pass its electrons to coenzyme Q, which will be use in the electron transport chain.

  His-FAD + succinate <--> His- FADH2 + fumarate

7. Hydration of fumarate to malate: fumarate is then converted to malate by using fumarase. This enzyme is highly stereospecific; it catalyzes hydration of the trans double bond of fumarate. The reaction with add H+ and OH- to make L-malate.

  Fumarate + H2O --> L-Malate

8. In the final step, Malate (C4), in a oxidation-reducation reaction catalyzed by malate dehydrogenase, is oxidized to oxaloacetate (C4) with NAD+ being reduced to NADH. The reaction is very positive with ΔG° = +29.7 kJ mol -1.

  Malate + NAD+ <--> oxaloacetate + NADH + H+

9. Then, the Krebs cycle restarts again as long as Oxygen is transported into the cell.

Note that the number of Carbon is going from the sum of 4 and 2 from the beginning to 6, 6, 5, 4, 4, 4, and back to oxaloacetate (C4) again. Overall, there are 6 NADH, 2 FADH2, and 4 CO2 being produced by two acetyl-CoA molecules from the Pyruvate Dehydrogenase Complex.

Lipoamide Between Different Active Sites edit

Since all the complex pyruvate dehydrogenase structure is known, the atomic model was able to be formed so that its activity could be understood from it. The center of the complex is formed by the transacetylase component E2. Transacetylase contains eight catalytic trimers which are gathered to form a hollow cube. There are three major domains for each three subunits that form a trimer. The amino terminus has a small domain that contains a bound flexible lipoamide cofactor which is attached to a lysine residue. This domain is homologous to biotin-binding domains like pyruvate carboxylase. The lipoamide domain is followed by a small domain that interacts with E3 within the complex and the larger transacetylase domain completes an E2 subunit. E1 is considered to be α2β2 tetramer, and E3 is considered to be an αβ dimer. E2 is surrounded by multiple copies of E1 and E3. These three distinct active sites work together through the long, flexible lipoamide arm of the E2 subunit that carries substrates from one active site to the other. The process of moving lipoamide between different active sites are:
1) In the active site of E1, pyruvate is decarboxylated and forms a hydroxyethyl-TPP intermediate while the CO2 leaves as the first product. The active site is connected to the enzyme’s surface through a long hydrophobic channel within the E1 complex.
2) The lipoamide arm of the lipoamide is inserted by E2 into the deep channel in E1 which leads to the active site.
3) The transfer of the acetyl group is catalyzed by E1 to the lipoamide. The acetylated arm leaves E1 and enters the E2 cube in order to visit the active site of E2. This is located deep in the cube at the subunit interface.
4) The acetyl moiety is then transferred to CoA. The second product which is the acetyl CoA, leaves the cube. The reduced lipoamide arm then swings to the active site of the E3 flavoprotein.
5) The lipoamide is oxidized by the coenzyme FAD in the E3 active site. The reactivated lipoamide is ready to start a new reaction cycle.
6) NADH, the final product, is produced through the reoxidation of FADH2 to FAD.

This coordinated catalysis of a complex reaction is possible because of the structural integration of three different kinds of enzymes and the long, flexible lipoamide arm. The overall reaction rate is increased and the side reaction is minimized due to the proximity of one enzyme to another. Throughout the reaction sequence, all the intermediates in the oxidative decarboxylation of pyruvate remain bound to the complex. This is readily transferred to the flexible arm of E2 calls on each active site in turn.

Pyruvate Dehydrogenase Complex edit

Although glucose can be formed by pyruvate, the irreversible step of the formation of acetyl CoA from pyruvate causes the acetyl CoA to be unable to convert back into glucose. The oxidative decarboxylation of pyruvate to acetyl CoA commits the carbon atoms of glucose to one of two principal fates: oxidation to CO2 by the citric acid cycle, with the concomitant generation of energy, or incorporation into lipid. The activity of the pyruvate dehydrogenase complex is stringently controlled. The reaction can be inhibited by the high concentration of reactions: by binding directly, the acetyl CoA inhibits the transacetylase component E2 while the NADH inhibits the dihydrolipoyl dehydrogenase E3. High concentrations of NADH and acetyl CoA inform the enzyme that the energy needs of the cell have been met or in order to produce the acetyl CoA and NADH, the fatty acids are being degraded to produce because most pyruvate is derived from glucose by glycolysis. Covalent modification is very important in regulating the complex in eukaryotes. The phosphorylation of the of the pyruvate dehydrogenase component (E1) by pyruvate dehydrogenase kinase I (PDK) switches of the activity of the complex. The deactivation is reversed by the pyruvate dehydrogenase phosphate.

Regulatory Enzyme in Citric Acid Cycle edit

In animal cells, the rate of citric acid cycle is regulated to fitted the required needs for ATP. The two enzymes that allosteric control the cycle are isocitrate dehydrogenase and α-ketoglutarate dehydrogenase, which both generate high-energy electrons in cycle.

Isocitrate dehydrogenase is stimulated by ADP that increase the affinity of enzyme for substrates. Since isocitrate, NAD+, Mg+, and ADP are cooperative, the binding of these substrates are regulated. If energy is unnecessary, there will be more NADH and ATP that will compete for the binding of enzyme with NAD+ and ADP respectively. Because the reaction requires positive energy or electron acceptors, the reactions is slowed until high energy like ATP and NADH are needed.

α-ketoglutarate dehydrogenase is another allosteric enzyme that regulated the rate of citric acid cycle for ATP. This enzyme is inhibited by succinyl CoA and NADH, which are the product that also compete for binding with reactants. α-ketoglutarate is also inhibited by high energy electron.

Mitochondrial electron transport chain

Definition edit

Between mitochondrial matrix and intermembrane space, within the innermembrane, there are 5 complexes involved in the oxidative phosphorylation cycle, also known as electron transport chain cycle

  1. At complex I - NADH dehydrogenase: NADH is oxidized to NAD+: a process that releases electrons, which is transported by FMN (flavin mononucleotide to the Fe-S center (similar to heme group of hemoglobin) and reduce Q (ubiquinone) to QH2 (ubiquinol). Besides, 4 protons are pumped from matrix to intermembrane space.
  2. At complex II - Succinate dehydrogenase: succinate is oxidized to fumarate: a process that releases electrons to reduce FAD to FADH2, which carries the original electrons to reduce Q (ubiquinone) to QH2 (ubiquinol). Again protons are pumped into the intermembrane space
  3. At complex III - Ubiquinone Cytochrome c oxidoreductase: Q-cycle transfers electron from QH2 to cytochrome c (cyt c).
  4. At complex IV - cytochrome oxidase: cyt c transfer electrons to oxygen O2 (by the support of Cu-S center and hemes). This is where water is released and proves the role of oxygen in aerobic respiration. Again protons are pumped into the intermembrane space
  5. At complex V - ATP Synthase: with protons pumped into intermembrane space now return to the matrix, a rotary complex carries out the job of combining ADP and inorganic phosphate in mitochondria into ATP for cellular energy.
Atp synthase

ATP Synthase edit

While the electron transport chain transports electrons and pumps H+ ions into the intermembrane space, the process of ATP synthesis does not occur until the ATP Synthase. The other part of the oxidative phosphorylation, after the electron transport chain, is the ATP synthase. ATP synthase is a transport protein that is consists of four parts, the Stator, the Rotor, the internal rod, and the Catalytic knobs.

  1. The H+ ions in the intermembrane space pumped by the electron transport chain will flow down their gradient first through the stator. The stator is anchored in the membrane.
  2. The H+ ion then binds onto the rotor, which is shaped somewhat like a waterwheel. This binding causes the rotor to change its shape and thus, makes it spin within the membrane.
  3. The spinning of the rotor causes the internal rod to spin, which leads the catalytic knob to spin. The spinning of the catalytic rod causes the catalytic sites in the rod to transform ADP and inorganic phosphate group into ATP in the mitochondrial matrix.

Overall, the ATP synthase functions like a waterwheel. When the concentration of H+ ions in the intermembrane space becomes higher than that of the matrix, the H+ ions will go down the ATP synthase and facilitate the rotor of the ATP synthase. This causes the other components of the ATP synthase to spin. As a result, ATP can be generated from the catalytic sites in the catalytic rod.

Chemiosmosis edit

Chemiosmosis is the movement of ions across a membrane down its gradient. It couples the electron transport chain and chemiosmosis in order to create ATP. In electron transport chain, the multiprotein structure pumps out H+ ions into the intermembrane space. As the H+ ions get pumped out, the concentration of H+ in the intermembrane space gets higher. As a result, H+ ions will start flowing down back to the chromosomes matrix through the ATP molecule. Through this movement, the cardiac rotor of the ATP synthase transforms the ADP into ATP.

Background Information edit

Gluconeogenesis pathway with key molecules and enzymes. Many steps are the opposite of those found in the glycolysis.

Gluconeogenesis is a process, in which Pyruvate (a product of Glycolysis) is backward-converted into sugar, glucose in particular. Which can then be stored in the form of glycogen in animals' cells or starch and cellulose in plants' cells.

Process edit

There are three basis steps involved in Gluconeogenesis:

  1. Pyruvate to Phosphoenolpyruvate (PEP):
    1. The enzyme pyruvate carboxylase converts pyruvate into oxaloacetate by adding the CO2 from the bicarbonate ions with the support of ATP and the coenzyme biotin.
    2. Then oxaloacetate is converted into phosphoenolpyruvate (PEP)by the enzyme PEP carboxykinase in a process that uses GTP (guanosine triphosphate) as energy and releases CO2 as a waste product. Note that the released CO2 is actually the same CO2 molecule from the bicarbonate ion at the previous step.
  2. The next steps are just the reversed processes of glycolysis going from PEP back to Fructose 1,6-bisphosphate, with released products in glycolysis being reactants/cofactors in gluconeogenesis.
  3. Fructose 1,6-bisphosphate (Fru 1,6-P) to Fructose 6-Phosphate (Fru 6-P): This is an irreversible hydrolysis reaction catalyzed by the enzyme Fructose 1,6-bisphosphatase (FBPase-1). This reaction is heavily regulated by Fructose 2,6-bisphosphate.
  4. Glucose 6-Phosphate to Glucose: Fru 6-P in the previous step is converted into Glucose 6-Phosphate by reversed step of glycolysis. Glucose 6-phosphate is converted into glucose by a hydrolysis reaction.

Regulation edit

The main regulatory factor is the concentration of Fructose 2,6-bisphosphate (Fru 2,6-P), which controls glycolysis (catabolic pathway) and gluconeogenesis (anabolic pathway).

  1. First, Fru 2,6-P is the conversion of Fructose 6-Phosphate (Fru 6-P) by the enzyme Phosphofructokinase-2 (PFK-2), which uses ATP as an energy source. This is a reversible reaction, thus the reversed reaction gives back Fruc 6-P. The reversed reaction is catalyzed by the enzyme Fructose 2,6-bisphosphatase (FBPase-2) in a process releasing an inorganic phosphate.
  2. Second, how can Fru 2,6-P regulates glycolysis and gluconeogenesis?
    1. In a breakdown pathway, because Fru 6-P is readily phosphorylated into Fru 1,6-P by the enzyme Phosphofructokinase-1 (PFK-1), a high concentration of Fru 2,6-P will accumulate and promotes the activity of PFK-1 and inhibits FBPase-1. Note that PFK-2 is active and FBPase-2 remains inactive to stimulate the production of Fru 2,6-P
    2. In the synthesis pathway, Fru 6-P concentration rises, inhibits the activity of PFK-2, and stimulates FBPase-2 to reduce the concentration of Fru 2,6-P. As the concentration of Fru 2,6-P decreases over time, the activity of PFK-1 also decreases, thus stimulating FBPase-1 to convert Fru 1,6-P into Fru 6-P,which is later ultimately converted into glucose.

Note that in the case of low glucose level in the blood: the enzyme glucagon from liver can stimulate the production of an enzyme called cAMP-dependent protein kinase, which stimulates the conversion of the PFK-2 (active)/FBPase-2 (inactive) into PFK-2 (inactive)/FBPase-2 (active). This process will inhibit glycolysis and promote gluconeogenesis to pump more glucose from liver into the blood.


Overview edit

Phosphofructokinase-1 (PFK-1) is a major factor in process known as glycolysis. PFK-1 catalyzes the committed step, or begins an irreversible enzymatic reaction, which is the point of no return. PFK-1 also regulates triacylglycerol synthesis, which helps decrease the amount of fat storage. By preventing PFK-1 from doing its job, other glycolytic intermediaries decrease such as 2,3-bisphosphoglycerate.

Defective Phosphofructokinase-1 edit

Faulty PFK-1 can cause a build up of glucose, glucose-6-phosphate, and other glycolytic intermediaries. The end result of this is glycogen synthesis, which can lead to increased solute concentration in red blood cells, enlarged spleen, a swelled heart, and ultimately death.

References edit

1. "Phosphofructokinase-1." Phosphofructokinase-1. N.p., n.d. Web. 07 Dec. 2012.

PFK-1 edit

Phosphofructokinase-1 (PFK-1) is a putative controlling enzyme in glycolysis. Cancer cells have defective mitochondria, which forces them to depend on glycolysis as their main source of energy. Ascorbate (commonly known as Vitamin C) has been shown to inhibit PFK-1 and Lactate Dehydrogenase (LDH). In the muscle cells, when the muscle is active, PFK-1 and LDH form a complex with contractile proteins in the muscle, protecting them from inhibition allowing for glycolysis (glucose is degraded and ATP is made. When the muscle is at rest, it is hypothesized that the complex is not formed, and PFK-1 and LDH are inhibited by ascorbate, glycolysis does not occur and glucose is stored as glycogen. Glycogen storage mainly occurs in the liver and muscle tissue. It is also hypothesized that because cancer cells depend mainly on glycolysis for energy, that PFK-1 in cancer cells will not be inhibited by ascorbate. In order to test this hypothesis it is important to be able to purify PFK-1. Because aldolase (Ald) protects from inhibition, the purified PFK-1 must not have aldolase in it in order to properly test the hypothesis.

Micro-purification Method edit

Rabbit muscle was used as a source of PFK-1. Preparation of three assays (PFK-1 assay, LDH assay, and aldolase assay) were necessary to test the enzyme activity after each step using a spectrophotometer. This is done to keep track of what is removed and what stays after each step.

Rabbit muscle was blended in a solution of ethylenediaminetetraacetic acid (EDTA), dithiothreitol (DTT, and sodium fluoride (NaF), then centrifuged and washed in order to remove soluble proteins. This was done multiple times to try and remove some LDH and aldolase.

Heat Step The pellet containing the PFK-1 was then resuspended in a solution of Tris, MgSO4, EDTA, ATP, and DTT and then submerged in a heat bath and then placed in an ice bath. This was done to denature the other proteins while solubilizing and keeping safe the PFK-1 by using ATP and magnesium. The suspension was centrifuged and the supernatant saved.

Ion-exchange and Dye Columns
The supernatant was then placed on four different columns, all standardized with 40TP8 and DTT. The four columns were: blue agarose, brown agarose, blue dextran, and DEAE-Sephacel. The supernatant was allowed to drip through the columns by gravity and then collected to be tested. Theoretically, the PFK-1 should be stuck to the columns at that point. Solutions of 40TP8/ DTT were then added to the columns and collected in fractions. And then solutions containing an increased concentration of TP8 were added and then collected in fractions. The purpose of adding increasing concentrations of TP8 was to wash off any other proteins with a weaker affinity for the beads in the columns first. An additional method using a vacuum filtration system was tried, but would pull the PFK-1 and aldolase through the column too quickly, not giving PFK-1 enough time to stick to the column, making this method counterproductive.

Gel Electrophoresis Samples taken from the column were then tested for purity by polyacrylamide gel electrophoresis (PAGE)

Results and Discussion edit

The DEAE Sephacel column seemed to have the highest yield of PFK-1 activity out of the four columns, but when tested using PAGE there was a lot of contamination. As mentioned before, there shouldn’t be aldolase because it inhibits PFK-1, which would make testing the affects of ascorbate on PFK-1 very hard to do. The blue agarose, while it didn’t have as high of a yield as DEAE Sephacel, showed to be very pure when tested by PAGE. The brown agarose showed potential at producing high yields of PFK-1, but after continuous testing, while it was good at removing aldolase, the percent yield of PFK-1 was too low.
Blue agarose so far has shown to be the best choice out of the four, but continuous testing/adjusting must be done before using this process on cancerous tissue. This is because while blue agarose removes aldolase well, the yield is still not high enough for it to be efficient to use with cancer tissue.

References edit

1. Queja, A., Marshall, A., Willaims, A., & Russell P.J. (2012, June). Micro-purification of Rabbit Phosphofructokinase-1. Poster presented at the biennial symposium of the Intercultural Cancer Council (ICC), Houston, TX.

2. Russell P, Williams A, Marquez K, Tahir Z, Hossein B, Lam K. 2008. Some characteristics of rabbit muscle phosphofructokinase-1 inhibition by ascorbate. J Enzyme Inhib & Med Chem; 23: 411-417

3. Vassault, A. 1983. Methods of Enzymatic Analysis, Enzymes I: Oxidoreductases, Transferases Vol. III, Verlag Chemie, Basel, pp. 118–126.

Overview edit

Light Reaction converts light to ATP and NADPH, an electron carrier. This reaction occurs in the thylakoid, and it requires visible light for the chlorophyll (a type of molecule known as pigment that absorbs light) to absorb all visible light except for green. Chlorophyll are organized in the thylakoid membrane (in the photosystems).

Then the electron gains energy from light and jumps up to a higher electron shell; this excited state is unstable and temporary, and so it jumps back to the original position. As it returns to its ground state, it gives off energy in the form of heat, and this heat is then absorbed by a neighboring chlorophyll, which passes on the heat to another neighboring chlorophyll and so on until the heat reaches the reaction center chlorophyll. At this point, the reaction center chlorophyll's electron gets excited and passed on to the primary electron acceptor, and the reaction center chlorophyll results with a missing electron that is regained from H2O (electron donor). Before this stage, H2O is converted to 2e- + 2H + 1/2 O2 and this is how it is able to donate an electron. The primary electron acceptor now has an extra electron, which is donated to the electron transport chain that has H+ pumping. The ATP synthase pumps the H+ back in to the stroma and converts ADP + P → ATP. All of this occurs in Photosystem II. Then, the final electron acceptor is the reaction center chlorophyll of PhotoSystem I. PS I receives an electron from PS II, and the primary electron acceptor of PS I donates the electron to another electron transport chain. This electron transport chain has no H+ pumping and no ATP; instead, it generates NADPH from NADP+. Thus, the purpose of the light reaction is to convert light to chemical energy in the form of ATP and NADPH [3]

Photosystem II and I

There are three steps in converting Light energy to ATP and NADPH

  1. To capture the light energy from the sun
  2. Undergo Photosystem II and I to convert ATP and NADPH
  3. The converted ATP and NADPH is used to reduce CO2 to sugar

The photosystems undergo in the internal membrane called Thylakoid membrane. Thylakoid membrane is membrane full of collections of pigments and proteins. There are four types of pigments they are: chlorophylls, carotenes, xanthophylls and phycobilins. The light reaction uses the light power to create ATP and NADPH2 to provide reducing energy to undergo the chemical reaction to reduce the CO2 to sugar. [4]

References edit

  1. Berg, Jeremy M. (2010). Biochemistry (7th Ed. ed.). W. H. Freeman and Company. ISBN0-1-42-922936-5. {{cite book}}: |edition= has extra text (help)
  2. Berg, Jeremy M. (2010). Biochemistry (7th Ed. ed.). W. H. Freeman and Company.