title: Protein disulfide-isomerase interacts with soluble guanylyl cyclase via a redox-based mechanism and modulates its activity.
abstract: 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.
authors.affiliation: Department of Pharmacology and Physiology, New Jersey Medical School, UMDNJ, Newark, NJ 07103, USA. heckleer@umdnj.edu
authors.forename: Erin J
authors.initials: EJ
authors.lastname: Heckler
authors.forename: Pierre-Antoine
authors.initials: PA
authors.lastname: Crassous
authors.forename: Padmamalini
authors.initials: P
authors.lastname: Baskaran
authors.forename: Annie
authors.initials: A
authors.lastname: Beuve
mesh_terms: D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D004789:Enzyme Activation / Q000502:physiology; D006162:Guanylate Cyclase / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D051379:Mice; D010084:Oxidation-Reduction; D011485:Protein Binding / Q000502:physiology; D019704:Protein Disulfide-Isomerases / Q000737:chemistry / Q000378:metabolism*; D025941:Protein Interaction Mapping / Q000379:methods; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry / Q000378:metabolism*; D000071756:Soluble Guanylyl Cyclase
chemical_list: D018160:Receptors, Cytoplasmic and Nuclear; D006162:Guanylate Cyclase; D000071756:Soluble Guanylyl Cyclase; D019704:Protein Disulfide-Isomerases