Structural Biochemistry/Cell Signaling Pathways/Nitric Oxide and CGMP Response

Nitric oxide (NO) plays an important role in the biological system as a vital signaling molecule. In mammalian physiology, this gaseous molecule functions as the primary activator of soluble guanylate cyclase (sGC) in the cyclic guanosine monophosphate (cGMP) pathway. When coupled with enzyme nitric oxide synthase (NOS), synthesis of NO is derived from L-arginina and oxygen (O2). NO binds to the heme cofactor of SGc after diffusing across the cell membrane. From this, sGC can only form a stable structure with NO and carbon monoxide (CO), but not with O2. The binding of sGC with NO results in a substantial increase in cGMP levels in the system. The second messenger then modifies phosphodiesterases (PDEs), gated-ion channels, or cGMP-dependent protein kinases to maintain physiological tasks , such as platelet aggregation, vasodilation, and neurotransmission. In pursuit of therapeutic intervention in diseases concerning the NO/cGMP-sginaling pathway, many studies have been centered on the explication of sGC activation/deactivation. This article condenses the contemporary knowledge of sGC form and function as well as recent works in NO signaling.

Following the discovery of the nitric oxide/cyclic guanosine monophos-phate (NO/cGMP) pathway in the 1980s, cGMP production has stimulated with the clinical administration of organic nitrites, specifically glycerol trinitrate (GTN). These compounds relieve the pain following angina by soothing vascular smooth muscle, leading to vasodilation. During the past years, studies concerned the mechanism of smooth muscle relation by these compounds, which gave rise to the discovery of NO as a signaling molecule. In addition, this also led to the discovery of the enzymes that synthesize NO and cGMP.

It has been shown that both cytosolic and particulate fractions of mammalian tissue exhibit guanylate cyclase activity. Within these particulate fractions are membrane-bound particulate guanylate cyclases that are activated by natriuretic peptides (reviewed in References 3 and 4). In contrast, cytosolic fractions contain soluble guanylate cyclases (sGCs) that are activated by NO. NO-responsive guanylate cyclase activity is also exhibited within the cell membranes of certain tissues, such as skeletal muscle and brain, as well as in platelets (5-7). Most tissues contain Guanylate cyclases, while the protein distribution in these tissues are isoform specific. Because localized groups of the signaling compound can be synthesized within specific types of tissues and in closeness to either soluble or membrane bound cGMP receptors, this provides another reason to regulate cGMP-dependent responses. Therefore, specific tissues can control cGMP levels by expressing unique GC isoforms, which have distinct peptide receptors/ligand activators. In addition, during human and mouse vascular homeostasis (8), a reciprocal communication between particulate guanylate cyclase and sGC has been. It is likely that communication between these two pathways is performed through several processes involving cGMP. In eukaroytic individuals, NO signaling is marked by the initial release of calcium, then the binding of a calcium/calmodulin complex to nitric oxide synthase (NOS), which causes the enzyme to be activated. Then, following NO synthesis, it diffuses into target cells and binds to the heme in sGC. sGC is a histidine-ligated hemoprotein that binds NO and carbon monoxide (CO), but not oxygen (O2). As a result, cGMP synthesis increases several hundredfold.

Over time, development of a cost-effective technique for cGS purification has progressed little, but several techniques have been developed to yield about a microgram of the homogeneous product. Primarily, cGS extraction derived from rat and bovine tissues. In the 1980s, purifies sGC for studies was obtained from rat lung, liver as well as bovine lung. Regardless, it was observed that sGC could be purified without the heme cofactor, depending on the purification method. The use of ammonium sulfate precipitation and solubilizing agents can lead to misfolding protein synthesis. Heme reconstruction of this misfiled protein produces a unique sGC that is biochemically different from the native protein. Currently, the bovine lung sGC method is the most effective and efficient method of isolating heme-bound protein from source tissue. For one kilogram of lung, these is a 1 mg protein yield. In further advancement, sGC production advanced from the progress of heterologous expression systems for recombinant sGC expression. COS-7 cells was used for the first successful heterologous expression system. The establishment of sGC as an obligate heterodimer containing both alpha 1 and beta 1 subunits was achieved despite the low sGC from COS-7 cells. In addition, COS-7 cells were used to examine truncations and mutants of sGC through lysate activity assays. The first procedure to separate pure recombinant protein was the overexpression of ratsGC inside insect cells with the Sf9/baculovirus expression system. sGC expression in insect cells became successful without an affinity tag, but recent techniques require a His tag to accomplish the purification process. Most of the protein is insoluble. From this method, there is a 0.2-0.4 mg yield of pure soluble protein per liter of culture. Up to date, this is now commonly used to obtain purified rat and human of pure protein per liter of culture. Another method using an E. coli expression system was used for heterodimer, Manduca sexta. It produces (0.5-1.0 mg/liter) of only partially pure protein of full length. Higher yield were obtained using the truncation of the C terminus of the alpha one and beta one subunits. Although the resulting heterodimeric proteins cannot cyclize GTP, they can still be purified to homogenitiy.

The structure of heterodimeric sGC contains two homologous subunits, alpha and beta. Most studies focus on the isoform, alpha-one beta-one protein although alpha-two and beta-two subunits have also been discovered. Initially, these proteins existed only in mammals, but also in insects like Drosophila melanogaster and M. sexta, and in fish. In mammals, humans, rats and cows, the localization of the alpha and beta subunit has been studied. By techniques of Western blotting and quantitative polymerase chain reaction analysis, the alpha-two subunit is less available than the alpha-one and beta-one counterparts. It is highly present in the brain, colon, heart, spleen lung, placenta and unteres. Studies have shown that with purified protein, the alpha-two beta-one heterodimer has ligand-binding character that is completely similar the alpha-one beta-one heterodimer, but a spliced variant of the alpha-two subunit combines to form a dimer with the beta-one subunit to make an chemically inactive complex.

Currently, several studies in mice show the importance of various sGC isoforms for physiological function. It seems that the mice lacking the sGC beta-one subunit displayed high blood-pressure, low heart rate and gastrointestinal contractility disorder. In addition, removal of the beta-one subunit within smooth muscle cells causes loss of the protein in these cells like the hypertension in the knockout mice. In general, deletion of the beta-one subunit tends to be viewed as a global sGC knockout because the alpha-one and beta-one are not compatible heterodimers with beta-two.sGC alpha-one and alpha-two knockout mice were also made. Here, both proteins were discovered to be vital for long-term potentiation, and vasodilation, the contraction of blood vessels in smooth muscle tissue. Studies have shown that the alpha-one subunit-deficient mice have had both alpha-subunits to be participation of colon tissue.

Reference

Structure and regulation of soluble guanylate cyclase. Derbyshire ER, Marletta MA. Annu Rev Biochem. 2012;81:533-59. Epub 2012 Feb 9. Review.