S-Glutathiolation by peroxynitrite activates SERCA during arterial relaxation by nitric oxide

Abstract

Nitric oxide (NO) physiologically stimulates the sarco/endoplasmic reticulum calcium (Ca(2+)) ATPase (SERCA) to decrease intracellular Ca(2+) concentration and relax cardiac, skeletal and vascular smooth muscle. Here, we show that NO-derived peroxynitrite (ONOO(-)) directly increases SERCA activity by S-glutathiolation and that this modification of SERCA is blocked by irreversible oxidation of the relevant cysteine thiols during atherosclerosis. Purified SERCA was S-glutathiolated by ONOO(-) and the increase in Ca(2+)-uptake activity of SERCA reconstituted in phospholipid vesicles required the presence of glutathione. Mutation of the SERCA-reactive Cys674 to serine abolished these effects. Because superoxide scavengers decreased S-glutathiolation of SERCA and arterial relaxation by NO, ONOO(-) is implicated as the intracellular mediator. NO-dependent relaxation as well as S-glutathiolation and activation of SERCA were decreased by atherosclerosis and Cys674 was found to be oxidized to sulfonic acid. Thus, irreversible oxidation of key thiol(s) in disease impairs NO-induced relaxation by preventing reversible S-glutathiolation and activation of SERCA by NO/ONOO(-).

Cardiac contractile dysfunction in Lep/Lep obesity is accompanied by NADPH oxidase activation, oxidative modification of sarco(endo)plasmic reticulum Ca2+-ATPase and myosin heavy chain isozyme switch

“…Oxidative stress has been shown to result in oxidative damage to functional proteins [35]. SERCA2a has been shown to be a vulnerable target for oxidative insult [27,36]. In this study we demonstrated a marked oxidative modification of SER CA2a in Lep/Lep mouse myocytes, which may contribute to reduced SERCA activity and subsequent cardiac contractile dysfunction in Lep/Lep mouse myocytes.…”

“…Filters were then removed from the manifold, placed in scintillation fluid and counted. SERCA activity was expressed as cpm/mg protein [27].…”

“…8). Effect of obesity on SERCA2a oxidation and SERCA activity Persistent oxidative stress leads to oxidative damage to proteins such as SERCA [27,35,36]. Given that protein carbonyl formation, a fundamental measure of oxidative damage in cardiac proteins [22,37], is elevated in Lep/Lep heart, we evaluated oxidative damage to SERCA2a by quantitative immunoprecipitation of SERCA2a from cell lysates using specific SERCA2a antibody followed by immunoprobe of the protein carbonyl content.…”

Redox-based regulation of signal transduction: Principles, pitfalls, and promises

“…However, even in vascular smooth muscle, a significant component of the relaxation response may be cGMP independent. In some vessels and species, the response may even be completely independent of cGMP [5][6][7][8], and it has been shown that animals deficient in a SNO-metabolizing enzyme have low vascular resistance ( [9], JSS unpublished). By analogy, stringent genetic evidence for a role of NO (per se) in blood pressure control has not been provided.…”

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

“…However, although these studies suggested peroxynitrite was responsible for the S-nitrosation of SERCA they did not measure disulfide formation [41]. Prior to these S-nitrosation-focused studies, peroxynitrite was shown to directly increase SERCA-dependent Ca 2+ uptake activity due to a disulfide, namely S-glutathiolation of Cys-674 [118]. During atherosclerosis Cys-674 was oxidised to a sulfonic acid, preventing Sglutathiolation and limiting SERCA activity, illustrating the functional importance of disulfide in the control of SERCA activity.…”