Nitric Oxide-induced S-Glutathionylation and Inactivation of Glyceraldehyde-3-phosphate Dehydrogenase

Abstract

S-Nitrosylation of protein thiol groups by nitric oxide (NO) is a widely recognized protein modification. In this study we show that nitrosonium tetrafluoroborate (BF 4 NO), a NO؉ donor, modified the thiol groups of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by Snitrosylation and caused enzyme inhibition. The resultant protein-S-nitrosothiol was found to be unstable and to decompose spontaneously, thereby restoring enzyme activity. In contrast, the NO-releasing compound S-nitrosoglutathione (GSNO) promoted S-glutathionylation of a thiol group of GAPDH both in vitro and under cellular conditions. The GSH-mixed protein disulfide formed led to a permanent enzyme inhibition, but upon dithiothreitol addition a functional active GAPDH was recovered. This S-glutathionylation is specific for GSNO because GSH itself was unable to produce protein-mixed disulfides. During cellular nitrosative stress, the production of intracellular GSNO might channel signaling responses to form protein-mixed disulfide that can regulate intracellular function. Nitric oxide (NO)1 is an important biological messenger that plays a role in physiological and pathophysiological conditions such as endothelium-dependent vasorelaxation, inflammation, and septic shock (1, 2). These multiple effects are based on its redox chemistry. NO can react with oxygen species and transition metals to form NO x , peroxynitrite (ONOO Ϫ ), and metal-NO adducts, respectively (3, 4). The interactions of NO with sulfhydryl-containing molecules and enzymes has gained considerable importance (5, 6). In many biological systems, nitrosation reactions transferring NO ϩ from a NO donor to a protein S Ϫ group affect protein function. Targets for this type of modification, among others, are bovine serum albumin (7), tissuetype plasminogen activator (8), gyceraldehyde-3-phosphate dehydrogenase (GAPDH) (9, 10), the N-methyl-D-aspartate receptor (11), oncogenic p21 ras (12), and transcriptional activators (13).The S-nitrosothiol of glutathione (GSNO) may be the most relevant biological molecule to carry out nitrosation reactions under physiological conditions (14 -16). It has been reestablished that the actions of the endothelium-derived relaxing factor more closely resemble a low molecular weight nitrosothiol rather than the NO ⅐ radical itself (17). However, S-nitrosothiols can decompose to form NO ⅐ and thiyl radicals (18), and the thiyl radical can lead to the production of protein-mixed disulfides also known as protein S-glutathionylation.In this study, we investigated the influence of NO donors on the glycolytic enzyme GAPDH which catalyzes the reversible oxidative phosphorylation of D-glyceraldehyde-3-phosphate by NAD ϩ and inorganic phosphate. GAPDH is comprised of four identical 37-kDa subunits. Each subunit contains four cysteines; two of them (Cys-149 and Cys-153) are located in the catalytic site of each GAPDH subunit. The catalytically active cysteine 149 interacts with a histidine to form a highly reactive thiolate group (cys-S Ϫ ) which is required for GAPDH...

Radical-free biology of oxidative stress

“…Reversible oxidation of the active site of protein tyrosine phosphatase-1B has been associated with control of biological function in redox signaling (39). Oxidation of Cys by S-glutathionylation (GS-ylation) regulates glyceraldehyde 3-phosphate dehydrogenase (125) and caspase-3 (173). Cys residues are present in active sites of detoxification enzymes such as glutathione transferases, cytochromes P-450, Trxs, and peroxiredoxins.…”

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

“…The impact of the residues surrounding a thiol was studied using GAPDH as a model. GAPDH Cys-149 was shown to be activated by an adjacent histidine, which deprotonates it to render it a stronger nucleophile, enabling cleavage of the S-N bond within GSNO resulting in either S-glutathiolation or S-nitrosation [19]. Pérez-Mato et al described the crystal structure of methionine adenosyl transferase and found Arg-357 and Asp-363 are in proximity to the active site Cys-131.…”

“…More recently GAPDH has also been described as a cardiac trans-nitrosylase in the heart, transferring NO between proteins [33]. However GAPDH is also regulated by disulfide formation at the very same Cys-150 site, for example undergoing both S-glutathiolation and S-nitrosation in response to GSNO [19]. Each of these modifications inhibited the dehydrogenase activity of GAPDH in vitro, but when only S-nitrosation was induced activity returned within 30 minutes -whereas inhibition by S-glutathiolation did not [108].…”

“…Although transient S-nitrosothiols are already acknowledged to be one mechanism by which disulfides form [19,20], it is important to reconsider and highlight such studies. This is because the vast majority of work relating to protein S-nitrosylation fails to recognise that in many cases, if not most, a disulfide is likely the predominant post-translational modification that mediates NOdependent alterations in function.…”

S-Glutathionylation of Ion Channels: Insights into the Regulation of Channel Functions, Thiol Modification Crosstalk, and Mechanosensing

“…For instance, in the case of enzymes such as glyceraldehyde phosphate dehydrogenase, cAMPdependent protein kinase, creatine kinase, and protein kinase C, the altered enzyme activities by S-glutathionylation could be detected using biochemical assays (16,35,53,60,79,96,130). For ion channels, patch-clamp electrophysiology is the gold standard to test ion channel function and Ca + + imaging is also commonly used to study a variety of Ca + + -permeable channels.…”