Since the discovery that the endothelium derived relaxing factor (EDRF) was the endogenous gas nitric oxide (NO), an astonishing number of physiological functions have been attributed to NO. Despite the widely recognized importance of NO, little is known about the mechanism of regulation of the NO receptor, the soluble guanylyl cyclase (sGC). sGC is a heme-containing heterodimer that catalyzes the formation of cGMP from the substrate GTP. Upon binding of NO to the heme, the sGC is activated several hundred fold. The sGC is a multi-domain signaling enzyme that contains the receptor-heme domain, a dimerization domain and the effector-catalytic domain. It is not known how the NO signal is propagated to the catalytic domain. The proposed studies seek to understand the structural and molecular basis of mechanisms of regulation of the sGC: 1) how the NO signal is transmitted from the receptor-heme domain to the catalytic-effector domain, 2) why sGC, following prolonged exposure to NO, becomes unresponsive to NO, 3) do endogenous modulators of sGC activity play a role in this inhibition. 1) Our initial structure-based mutational analysis of the sGC heme domain and dimerization domain (solved with our collaborator Dr. van den Akker) revealed specific regions in these domains that are crucial for NO signaling. We shall investigate how these mutants affect NO activation of sGC. Guided by our structural modeling and homology with ancient conserved domains, we also seek to probe the interactions between the sGC domains that are involved in sGC allosteric activation. 2) In our quest to understand the mechanism of desensitization of sGC, we discovered that sGC is S- nitrosylated in vitro and in vivo and that S-nitrosylation correlates with the loss of responsiveness to NO- stimulation, while the basal activity remains unaltered. S-nitrosylation is a post-translational modification in which a NO moiety is added to the free-thiol of specific cysteines. We will study the mechanism of desensitization of sGC by identifying and mutating the S-nitrosylated cysteines and analyze the resulting phenotypes. 3) Recent work from our laboratory and others suggests that there are endogenous modulators for the sGC. We have discovered that protein disulfide isomerase (PDI) inhibits NO-stimulated sGC activity. We will characterize the mechanism of inhibition and address its physiological relevance. Understanding the mechanisms of regulation of sGC and identifying regulatory molecules will be key to uncovering the molecular basis of and developing compensatory therapies for some types of hypertension, atherosclerosis and erectile dysfunction, which affect more than 60 million Americans.Nitric oxide (NO) induces in the blood vessels the production of a small molecule messenger cGMP which relaxes the vasculature. Dysfunction in the NO-cGMP pathway is responsible for many cardiovascular diseases including hypertension, erectile dysfunction and atherosclerosis which affect more than 60 million Americans. We seek to understand how the production of these molecules is controlled by the body.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM067640-08
Application #
7798644
Study Section
Special Emphasis Panel (ZRG1-CB-G (01))
Program Officer
Dunsmore, Sarah
Project Start
2003-04-15
Project End
2012-03-31
Budget Start
2010-04-01
Budget End
2011-03-31
Support Year
8
Fiscal Year
2010
Total Cost
$331,826
Indirect Cost
Name
University of Medicine & Dentistry of NJ
Department
Pharmacology
Type
Schools of Medicine
DUNS #
623946217
City
Newark
State
NJ
Country
United States
Zip Code
07107
Beuve, Annie (2017) Thiol-Based Redox Modulation of Soluble Guanylyl Cyclase, the Nitric Oxide Receptor. Antioxid Redox Signal 26:137-149
Huang, Can; Alapa, Maryam; Shu, Ping et al. (2017) Guanylyl cyclase sensitivity to nitric oxide is protected by a thiol oxidation-driven interaction with thioredoxin-1. J Biol Chem 292:14362-14370
Crassous, Pierre-Antoine; Shu, Ping; Huang, Can et al. (2017) Newly Identified NO-Sensor Guanylyl Cyclase/Connexin 43 Association Is Involved in Cardiac Electrical Function. J Am Heart Assoc 6:
Beuve, Annie; Wu, Changgong; Cui, Chuanlong et al. (2016) Identification of novel S-nitrosation sites in soluble guanylyl cyclase, the nitric oxide receptor. J Proteomics 138:40-7
Heckler, Erin J; Kholodovych, Vladyslav; Jain, Mohit et al. (2015) Mapping Soluble Guanylyl Cyclase and Protein Disulfide Isomerase Regions of Interaction. PLoS One 10:e0143523
Gonzalez, J Patrick; Crassous, Pierre-Antoine; Schneider, Joel S et al. (2015) Neuronal nitric oxide synthase localizes to utrophin expressing intercalated discs and stabilizes their structural integrity. Neuromuscul Disord 25:964-76
Duran, Walter N; Beuve, Annie V; Sanchez, Fabiola A (2013) Nitric oxide, S-nitrosation, and endothelial permeability. IUBMB Life 65:819-26
Ramachandran, Jayalakshmi; Schneider, Joel S; Crassous, Pierre-Antoine et al. (2013) Nitric oxide signalling pathway in Duchenne muscular dystrophy mice: up-regulation of L-arginine transporters. Biochem J 449:133-42
Fioramonti, Xavier; Deak, Adam; Deshpande, Srinidhi et al. (2013) Hypothalamic S-nitrosylation contributes to the counter-regulatory response impairment following recurrent hypoglycemia. PLoS One 8:e68709
Heckler, Erin J; Crassous, Pierre-Antoine; Baskaran, Padmamalini et al. (2013) Protein disulfide-isomerase interacts with soluble guanylyl cyclase via a redox-based mechanism and modulates its activity. Biochem J 452:161-9

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