NO binds to the receptor sGC (soluble guanylyl cyclase), stimulating cGMP production. The NO–sGC–cGMP pathway is a key component in the cardiovascular system. Discrepancies in sGC activation and deactivation in vitro compared with in vivo have led to a search for endogenous factors that regulate sGC or assist in cellular localization. In our previous work, which identified Hsp (heat-shock protein) 70 as a modulator of sGC, we determined that PDI (protein disulfide-isomerase) bound to an sGC-affinity matrix. In the present study, we establish and characterize this interaction. Incubation of purified PDI with semi-purified sGC, both reduced and oxidized, resulted in different migration patterns on non-reducing Western blots indicating a redox component to the interaction. In sGC-infected COS-7 cells, transfected FLAG-tagged PDI and PDI CXXS (redox active site ‘trap mutant’) pulled down sGC. This PDI–sGC complex was resolved by reductant, confirming a redox interaction. PDI inhibited NO-stimulated sGC activity in COS-7 lysates, however, a PDI redox-inactive mutant PDI SXXS did not. Together, these data unveil a novel mechanism of sGC redox modulation via thiol-disulfide exchange. Finally, in SMCs (smooth muscle cells), endogenous PDI and sGC co-localize by in situ proximity ligation assay, which suggests biological relevance. PDI-dependent redox regulation of sGC NO sensitivity may provide a secondary control over vascular homoeostasis.
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Research Article|
April 25 2013
Protein disulfide-isomerase interacts with soluble guanylyl cyclase via a redox-based mechanism and modulates its activity
Erin J. Heckler;
Erin J. Heckler
1
1Department of Pharmacology and Physiology, New Jersey Medical School, UMDNJ, Newark, NJ 07103, U.S.A.
1To whom correspondence should be addressed (email heckleer@umdnj.edu).
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Pierre-Antoine Crassous;
Pierre-Antoine Crassous
1Department of Pharmacology and Physiology, New Jersey Medical School, UMDNJ, Newark, NJ 07103, U.S.A.
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Padmamalini Baskaran;
Padmamalini Baskaran
2
1Department of Pharmacology and Physiology, New Jersey Medical School, UMDNJ, Newark, NJ 07103, U.S.A.
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Annie Beuve
Annie Beuve
1Department of Pharmacology and Physiology, New Jersey Medical School, UMDNJ, Newark, NJ 07103, U.S.A.
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Publisher: Portland Press Ltd
Received:
February 26 2013
Accepted:
March 11 2013
Accepted Manuscript online:
March 11 2013
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2013 Biochemical Society
2013
Biochem J (2013) 452 (1): 161–169.
Article history
Received:
February 26 2013
Accepted:
March 11 2013
Accepted Manuscript online:
March 11 2013
Citation
Erin J. Heckler, Pierre-Antoine Crassous, Padmamalini Baskaran, Annie Beuve; Protein disulfide-isomerase interacts with soluble guanylyl cyclase via a redox-based mechanism and modulates its activity. Biochem J 15 May 2013; 452 (1): 161–169. doi: https://doi.org/10.1042/BJ20130298
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