S-glutathionylation uncouples eNOS and regulates its cellular and vascular function

Abstract

Endothelial nitric oxide synthase (eNOS) is critical in the regulation of vascular function, and can generate both nitric oxide (NO) and superoxide (O2•−), which are key mediators of cellular signalling. In the presence of Ca2+/calmodulin, eNOS produces NO, endothelial-derived relaxing factor, from L-arginine (L-Arg) by means of electron transfer from NADPH through a flavin containing reductase domain to oxygen bound at the haem of an oxygenase domain, which also contains binding sites for tetrahydrobiopterin (BH4) and L-Arg1–3. In the absence of BH4, NO synthesis is abrogated and instead O2•− is generated4–7. While NOS dysfunction occurs in diseases with redox stress, BH4 repletion only partly restores NOS activity and NOS-dependent vasodilation7. This suggests that there is an as yet unidentified redox-regulated mechanism controlling NOS function. Protein thiols can undergo S-glutathionylation, a reversible protein modification involved in cellular signalling and adaptation8,9. Under oxidative stress, S-glutathionylation occurs through thiol–disulphide exchange with oxidized glutathione or reaction of oxidant-induced protein thiyl radicals with reduced glutathione10,11. Cysteine residues are critical for the maintenance of eNOS function12,13; we therefore speculated that oxidative stress could alter eNOS activity through S-glutathionylation. Here we show that S-glutathionylation of eNOS reversibly decreases NOS activity with an increase in O2•− generation primarily from the reductase, in which two highly conserved cysteine residues are identified as sites of S-glutathionylation and found to be critical for redox-regulation of eNOS function. We show that eNOS S-glutathionylation in endothelial cells, with loss of NO and gain of O2•− generation, is associated with impaired endothelium-dependent vasodilation. In hypertensive vessels, eNOS S-glutathionylation is increased with impaired endothelium-dependent vasodilation that is restored by thiol-specific reducing agents, which reverse this S-glutathionylation. Thus, S-glutathionylation of eNOS is a pivotal switch providing redox regulation of cellular signalling, endothelial function and vascular tone.

β3 Adrenergic Stimulation Restores Nitric Oxide/Redox Balance and Enhances Endothelial Function in Hyperglycemia

“…Glutathionylation, which involves formation of a reversible disulphide bond between glutathione and reactive cysteines on proteins with significant structural and functional consequences, is increasingly recognized in physiological and pathophysiological cell signaling 13. Of direct relevance to our objective, glutathionylation also mediates eNOS uncoupling in endothelial cells under oxidative stress 14. In contrast to uncoupling by depletion of the cofactor tetrahydrobiopterin (BH 4 ), which leads to O 2 •− generation in the oxidase domain of the enzyme instead of NO synthesis, uncoupling by glutathionylation of 2 highly conserved cysteine residues of eNOS leads to O 2 •− generation in the reductase domain and abolishment of NO production 14.…”

How widespread is stable protein S-nitrosylation as an end-effector of protein regulation?

“…This resulted in decreased synthesis of NO and potentiated superoxide production [127]. Additional regulation of endothelial NOS activity by disulfides was found when mass spectrometry analysis of aortas, which indicated that S-glutathiolation of Cys-689 and Cys-908 uncoupled endothelial NOS so that it generated superoxide instead of NO [106]. Thus NOS, like so many other enzymes reported to be Snitrosated, are also observed to form disulfides.…”

A molecular switch of “Yin and Yang”: S-glutathionylation of eNOS turns off NO synthesis and turns on superoxide generation

“…Increasing lines of evidence point to the important role of S-glutathionylation in the regulation of signaling and metabolic pathways in intact cellular systems. Indeed, Chen et al found that GSSG induced dose-dependent S-glutathionylation of human eNOS that was reversed by reducing agents β-mercaptoethanol or dithiothreitol [8] . S-glutathionylation of eNOS reversibly decreases NOS activity with a concomitant increase in ·O 2-generation primarily from the reductase domain.…”

“…Recently, Chen et al reported that S-glutathionylation of eNOS may be a unique mechanism for the redox regulation of eNOS [8] . It has been demonstrated previously that cysteine residues are critical for the maintenance of normal eNOS function [9] .…”