Redox Activation of Nox1 (NADPH Oxidase 1) Involves an Intermolecular Disulfide Bond Between Protein Disulfide Isomerase and p47 ^phox in Vascular Smooth Muscle Cells

Abstract

Objective— PDI (protein disulfide isomerase A1) was reported to support Nox1 (NADPH oxidase) activation mediated by growth factors in vascular smooth muscle cells. Our aim was to investigate the molecular mechanism by which PDI activates Nox1 and the functional implications of PDI in Nox1 activation in vascular disease. Approach and Results— Using recombinant proteins, we identified a redox interaction between PDI and the cytosolic subunit p47 phox in vitro. Mass spectrometry of crosslinked peptides confirmed redox-dependent disulfide bonds between cysteines of p47 phox and PDI and an intramolecular bond between Cys 196 and 378 in p47 phox . PDI catalytic Cys 400 and p47 phox Cys 196 were essential for the activation of Nox1 by PDI in vascular smooth muscle cells. Transfection of PDI resulted in the rapid oxidation of a redox-sensitive protein linked to p47 phox , whereas PDI mutant did not promote this effect. Mutation of p47 phox Cys 196, or the redox active cysteines of PDI, prevented Nox1 complex assembly and vascular smooth muscle cell migration. Proximity ligation assay confirmed the interaction of PDI and p47 phox in murine carotid arteries after wire injury. Moreover, in human atheroma plaques, a positive correlation between the expression of PDI and p47 phox occurred only in PDI family members with the a′ redox active site. Conclusions— PDI redox cysteines facilitate Nox1 complex assembly, thus identifying a new mechanism through which PDI regulates Nox activity in vascular disease.

Protein Disulphide Isomerase and NADPH Oxidase 1 Cooperate to Control Platelet Function and Are Associated with Cardiometabolic Disease Risk Factors

“…Since both PDI and Nox-1 are able to regulate ROS generation [ 12 ], we would anticipate them to be present in a similar location, and close to their site of activity. Indeed, PDI has been reported to be able to associate with p47phox, which is a cytosolic subunit that regulates the activation of Nox-1 [ 12 ]. Therefore, we initially assessed if PDI, p47phox and Nox-1 would co-localise in platelets using immunofluorescence microscopy ( Figure 1 ).…”

“…In vascular smooth muscle cells (VSMC), PDI has been shown to regulate the production of reactive oxygen species (ROS) through aiding the assembly of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 1 (Nox-1) [ 12 ]. Similar to PDI, Nox-1 has recently been shown to regulate platelet function [ 13 ].…”

“…Indeed, we have previously proposed that platelet PDI could be implicated in platelet hyperactivation found in obesity [ 20 ]. In addition, PDI expression has been shown to be elevated in atherosclerotic lesions [ 12 ], while Nox-1 is reported to be upregulated in mesenteric arteries of mice with metabolic syndrome and contributes to vasodilation of these vessels [ 21 ]. Despite evidence in vascular cells, the potential relationship between PDI and Nox-1 biochemistry and cardiometabolic risk factors has not been explored.…”

Oxidant-Resistant LRRC8A/C Anion Channels Support Superoxide Production by Nox1

“…Oher redox-reactive extracellular proteins, such as thioredoxin or protein disulfide isomerases (PDI), can also potentially regulate these disulfides. PDI has previously been shown to associate with and modulate Nox1 activity intracellularly (Janiszewski et al ., 2005), mediating Nox1 activation resulting by disulfide formation with p47phox, one of the cytoplasmic subunits of the oxidase (Gimenez et al ., 2019).…”

Protein disulfide isomerase in cardiovascular disease

“…/H 2 O 2 generation upon agonist stimulation (our unpublished work). The PDI-p47 phox interaction mediated by intermolecular disulfide bonds also occurs in vascular smooth muscle cells (VSMCs) and is required for NOX1 activation 69 . Thus, further studies are required to investigate the relationship between PDI and NOXproduced ROS.…”