Project description:The nitric oxide/soluble guanylyl cyclase (NO-sGC) signaling pathway regulates the cardiovascular, neuronal, and gastrointestinal systems. Impaired sGC signaling can result in disease and system-wide organ failure. This review seeks to examine the redox control of sGC through heme and cysteine regulation while discussing therapeutic drugs that target various conditions. Heme regulation involves mechanisms of insertion of the heme moiety into the sGC protein, the molecules and proteins that control switching between the oxidized (Fe3+) and reduced states (Fe2+), and the activity of heme degradation. Modifications to cysteine residues by S-nitrosation on the α1 and β1 subunits of sGC have been shown to be important in sGC signaling. Moreover, redox balance and localization of sGC is thought to control downstream effects. In response to altered sGC activity due to changes in the redox state, many therapeutic drugs have been developed to target decreased NO-sGC signaling. The importance and relevance of sGC continues to grow as sGC dysregulation leads to numerous disease conditions.
Project description:Genomic and behavioral investigations were performed to determine the effects of a mutation in a Drosophila soluble guanylyl cyclase gene. A mutant DGCalpha1[3] third chromosome was crossed into a natural rover (for[R]) or natural sitter (for[s]) genetic background. (See Osborne et al. 1997; PMID: 9242616.) First instar larvae were collected and grown on 60mm Petri plates containing 10 mL of food until mid-third instar. (Approximate density was 3 animals per mL food). Larvae were collected and washed quickly with distilled water and were flash frozen in liquid nitrogen. Co-reared larvae were tested for behavioural effects. Four independent collections were made for each of the two conditions (Rover_DGCalpha1[3] or sitter_DGCalpha1[3]). Keywords = Drosophila Keywords = foraging Keywords = behavior Keywords = cGMP Keywords = guanylyl cyclase Keywords = genetic background Keywords: other
Project description:Soluble guanylyl cyclase (sGC) is a key component of NO-cGMP signaling in mammals. Although heme must bind in the sGC β1 subunit (sGCβ) for sGC to function, how heme is delivered to sGCβ remains unknown. Given that GAPDH displays properties of a heme chaperone for inducible NO synthase, here we investigated whether heme delivery to apo-sGCβ involves GAPDH. We utilized an sGCβ reporter construct, tetra-Cys sGCβ, whose heme insertion can be followed by fluorescence quenching in live cells, assessed how lowering cell GAPDH expression impacts heme delivery, and examined whether expressing WT GAPDH or a GAPDH variant defective in heme binding recovers heme delivery. We also studied interaction between GAPDH and sGCβ in cells and their complex formation and potential heme transfer using purified proteins. We found that heme delivery to apo-sGCβ correlates with cellular GAPDH expression levels and depends on the ability of GAPDH to bind intracellular heme, that apo-sGCβ associates with GAPDH in cells and dissociates when heme binds sGCβ, and that the purified GAPDH-heme complex binds to apo-sGCβ and transfers its heme to sGCβ. On the basis of these results, we propose a model where GAPDH obtains mitochondrial heme and then forms a complex with apo-sGCβ to accomplish heme delivery to sGCβ. Our findings illuminate a critical step in sGC maturation and uncover an additional mechanism that regulates its activity in health and disease.
Project description:Genomic and behavioral investigations were performed to determine the effects of a mutation in a Drosophila soluble guanylyl cyclase gene. A mutant DGCalpha1[3] third chromosome was crossed into a natural rover (for[R]) or natural sitter (for[s]) genetic background. (See Osborne et al. 1997; PMID: 9242616.) First instar larvae were collected and grown on 60mm Petri plates containing 10 mL of food until mid-third instar. (Approximate density was 3 animals per mL food). Larvae were collected and washed quickly with distilled water and were flash frozen in liquid nitrogen. Co-reared larvae were tested for behavioural effects. Four independent collections were made for each of the two conditions (Rover_DGCalpha1[3] or sitter_DGCalpha1[3]). Keywords = Drosophila Keywords = foraging Keywords = behavior Keywords = cGMP Keywords = guanylyl cyclase Keywords = genetic background
Project description:The aberrant activation of the ERG oncogenic pathway due to TMPRSS2-ERG gene fusions is the major driver of prostate cancer initiation and progression. We identified the alpha1 and beta1 subunits of soluble guanylyl cyclase (GUCY1A1, GUCY1B1) as major ERG-regulated genes in prostate cancer cells. Soluble guanylyl cyclase (sGC) is the major mediator of nitric oxide signaling in cells that, upon nitric oxide binding, catalyzes the synthesis of cGMP and subsequently activates PKG. We showed in ERG-positive PCa cells (VCaP) that cGMP synthesis was significantly elevated by ERG, leading to increased PKG activity and cell proliferation. To further understand the functions of sGC-cGMP pathway in prostate cancer cells, we performed RNA-seq analyses in VCaP cells to identify genes that are regulated by sGC.
Project description:A new Dictyostelium discoideum cyclase gene was identified that encodes a protein (sGC) with 35% similarity to mammalian soluble adenylyl cyclase (sAC). Gene disruption of sGC has no effect on adenylyl cyclase activity and results in a >10-fold reduction in guanylyl cyclase activity. The scg- null mutants show reduced chemotactic sensitivity and aggregate poorly under stringent conditions. With Mn(2+)/GTP as substrate, most of the sGC activity is soluble, but with the more physiological Mg(2+)/GTP the activity is detected in membranes and stimulated by GTPgammaS. Unexpectedly, orthologues of sGC and sAC are present in bacteria and vertebrates, but absent from Drosophila melanogaster, Caenorhabditis elegans, Arabidopsis thaliana and Saccharomyces cerevisiae.
Project description:Soluble guanylate cyclase (sGC) is a heme-containing heterodimeric enzyme that generates many molecules of cGMP in response to its ligand nitric oxide (NO); sGC thereby acts as an amplifier in NO-driven biological signaling cascades. Because sGC helps regulate the cardiovascular, neuronal, and gastrointestinal systems through its cGMP production, boosting sGC activity and preventing or reversing sGC inactivation are important therapeutic and pharmacologic goals. Work over the last two decades is uncovering the processes by which sGC matures to become functional, how sGC is inactivated, and how sGC is rescued from damage. A diverse group of small molecules and proteins have been implicated in these processes, including NO itself, reactive oxygen species, cellular heme, cell chaperone Hsp90, and various redox enzymes as well as pharmacologic sGC agonists. This review highlights their participation and provides an update on the processes that enable sGC maturation, drive its inactivation, or assist in its recovery in various settings within the cell, in hopes of reaching a better understanding of how sGC function is regulated in health and disease.
Project description:Soluble guanylyl cyclase (GC-1) is the primary receptor of nitric oxide (NO) in smooth muscle cells and maintains vascular function by inducing vasorelaxation in nearby blood vessels. GC-1 converts guanosine 5'-triphosphate (GTP) into cyclic guanosine 3',5'-monophosphate (cGMP), which acts as a second messenger to improve blood flow. While much work has been done to characterize this pathway, we lack a mechanistic understanding of how NO binding to the heme domain leads to a large increase in activity at the C-terminal catalytic domain. Recent structural evidence and activity measurements from multiple groups have revealed a low-activity cyclase domain that requires additional GC-1 domains to promote a catalytically-competent conformation. How the catalytic domain structurally transitions into the active conformation requires further characterization. This review focuses on structure/function studies of the GC-1 catalytic domain and recent advances various groups have made in understanding how catalytic activity is regulated including small molecules interactions, Cys-S-NO modifications and potential interactions with the NO-sensor domain and other proteins.
Project description:Various newly prepared and previously known vitamin B?? derivatives have been studied as potential soluble guanylyl cyclase (sGC) activators. All compounds tested were found to activate the sGC enzyme, although to differing extents. The best results were obtained with the derivatives synthesized from c-lactone and possessing aliphatic amides in the c- and d-positions.
Project description:Soluble guanylyl/guanylate cyclase (sGC), the primary biological receptor for nitric oxide, is required for proper development and health in all animals. We have expressed heterodimeric full-length and N-terminal fragments of Manduca sexta sGC in Escherichia coli, the first time this has been accomplished for any sGC, and have performed the first functional analyses of an insect sGC. Manduca sGC behaves much like its mammalian counterparts, displaying a 170-fold stimulation by NO and sensitivity to compound YC-1. YC-1 reduces the NO and CO off-rates for the approximately 100-kDa N-terminal heterodimeric fragment and increases the CO affinity by approximately 50-fold to 1.7 microm. Binding of NO leads to a transient six-coordinate intermediate, followed by release of the proximal histidine to yield a five-coordinate nitrosyl complex (k(6-5) = 12.8 s(-1)). The conversion rate is insensitive to nucleotides, YC-1, and changes in NO concentration up to approximately 30 microm. NO release is biphasic in the absence of YC-1 (k(off1) = 0.10 s(-1) and k(off2) = 0.0015 s(-1)); binding of YC-1 eliminates the fast phase but has little effect on the slower phase. Our data are consistent with a model for allosteric activation in which sGC undergoes a simple switch between two conformations, with an open or a closed heme pocket, integrating the influence of numerous effectors to give the final catalytic rate. Importantly, YC-1 binding occurs in the N-terminal two-thirds of the protein. Homology modeling and mutagenesis experiments suggest the presence of an H-NOX domain in the alpha subunit with importance for heme binding.