Physiologically, the NO-GC1-cGMP signaling pathway is critically involved in vascular homeostasis via smooth muscle relaxation and in inhibition of platelet aggregation, and in synaptic plasticity in the nervous system. Pathophysiologically, dysfunction of this pathway is linked to increased risk of myocardial infarction and is involved in the development of atherosclerosis, hypercholesterolemia, hypertension, erectile dysfunction and diabetes mellitus (all characterized by impairment in vascular reactivity) as well as neurological disorders and retinal degeneration.
Structure Function analysis of GC1
The soluble Guanylyl Cyclase (sGC or GC1) is an heterodimer (α/β subunits) that contains the heme at which NO binds. In turn, GC1 activity is increased, producing high levels of cGMP. Despite recently solved Cryo-EM structures, the mechanism of NO activation is still poorly understood.
We discovered that GC1 is desensitized by S-nitrosation (addition of NO moiety to the free thiol of cysteine, Cys) and investigated the molecular mechanism by which S-nitrosation affects GC1 activity and the transduction of NO activation (left panel, below). We also found that GC1 forms a complex with thiol-redox proteins, protein disulfide isomerase (PDI) and thioredoxin 1 (Trx1) and that this association is thiol redox-based (middle panel). We further explored the role of the multiple and conserved Cys of GC1 and found that GC1 could potentially form disulfide bond, which could be part of the mechanism of activation (right panel).
Transnitrosation activity of GC1
More recently we discovered that GC1 once it is S-nitrosated can transfer its SNO group(s) to other proteins including the oxidoreductase Trx1, which, in turn, can transfer this SNO to other specific protein targets. This transnitrosation cascade was identified in vitro and in cells and is facilitated by nitrosative and oxidative stress conditions.
We have identified C73 of Trx1 as the recipient of the SNO group transfer from GC1. We have conducted a bioinformatics analysis of the transnitrosation targets of GC1, Trx1 and the GC1/Trx1 complex. Our next challenge is to determine the physiological and pathophysiological role of this transnitrosation cascade, using Cys knock-in of mouse models.
We use biochemistry, cell biology and integrative approaches to explore the mechanism of thiol-dependent modulation of GC1 and its involvement in vascular dysfunction under oxidative and nitrosative stress. Our current research areas are summarized in these questions:
• What is the molecular mechanism of thiol-based modulation of GC1? Are formation and breakage of disulfide bonds keys for the mechanism of activation?
• What is the function of the interaction between GC1 and thiol-redox proteins?
• What is the role of GC1 transnitrosation activity? Is it a rescue mechanism when GC1 is desensitized by S-nitrosation?
Our integrative physiological studies are conducted using CRISPR/Cas9 to create new mouse models (done by our Center Genome Editing at Rutgers), a new telemetry system allowing recording of blood pressure and cardiac functions in unrestrained mice, and intravital microscopy to assay dilation/relaxation of arterioles (mesenteric artery).
The approaches for structure functional analyses encompass, purification from insect cells infected with baculovirus constructs, mutational analysis, pharmacology to measure GC1 response to NO, biochemistry, and molecular and cell biology.
FUNDING
- National Institutes of Health, R01-GM112415 (PI) 09/20/2020-08/31/2024 (Competitive renewal of a R01 grant awarded in 2015). NO signaling by a Soluble Guanylyl Cyclase-Thioredoxin transnitrosation complex
2. National Institutes of Health, R01-GM067640 (PI) 08/01/2018-03/31/2022 (Competitive renewal of the R01 grant awarded since 2003). Regulation of soluble guanylyl cyclase, the NO-receptor
3. National Institutes of Health, R01-GM131092 (co-I) 09/10/2019–06/30/2023 (MPI: Sorgen and Harris). Mechanisms By Which Phosphorylation and Protein Partners Regulate Cx45.
A postdoctoral position is currently available to lead a project to integrate the molecular mechanisms of Nitric Oxide and cGMP signaling in vivo in the cardiovascular system of mouse models subjected to oxidative stress, using physiological and pharmacological approaches. The ideal candidate obtained his/her PhD recently and has expertise in cardiovascular biology.
Graduate students welcome too!
Annie Beuve's lab 