Glutathiolation of Proteins by Glutathione DisulfideS-Oxide Derived from S-Nitrosoglutathione

Abstract

S-Nitrosoglutathione (GSNO) undergoes spontaneous degradation that generates several nitrogen-containing compounds and oxidized glutathione derivatives. We identified glutathione sulfonic acid, glutathione disulfide S-oxide (GS(O)SG), glutathione disulfide S-dioxide, and GSSG as the major decomposition products of GSNO. Each of these compounds and GSNO were tested for their efficacies to modify rat brain neurogranin/RC3 (Ng) and neuromodulin/GAP-43 (Nm). Among them, GS(O)SG was found to be the most potent in causing glutathiolation of both proteins; four glutathiones were incorporated into the four Cys residues of Ng, and two were incorporated into the two Cys residues of Nm. Protein S-glutathiolation can be induced in cells by mild oxidative stress (1). GSSG has been shown to oxidatively regulate the activity of several purified enzymes including carbonic anhydrase III (2, 3), protein kinase C (PKC) 1 (4), human aldose reductase (5), and human immunodeficiency virus, type I protease (6), and in each case the effects of glutathiolation can be reversed by reducing agents. As the concentration of reduced GSH in the mammalian cells is in the millimolar range and that of GSSG is less than 5% of GSH, glutathiolation of proteins by GSSG in vivo is not likely an efficient mechanism. More recently, the superoxide-induced glutathiolation of protein (7) and that induced by peroxynitrite, nitric oxide (NO), and nitrosothiol, in particular, S-nitrosoglutathione (GSNO), are thought to be the main avenues leading to protein Sthiolation (8 -12). In mammalian cells, a relatively high concentration of GSH (0.5-10 mM) serves as an NO sink to form GSNO (13-16), which can undergo transnitrosylation with protein sulfhydryl group to form S-nitrosoprotein and GSH or to form protein-GSH mixed disulfide and nitroxyl (17)(18)(19)(20). GSNO can also release NO in the presence of cuprous ion (21), ascorbate (22), or thiols (20, 23) and serves as a possible source of nitrsonium or nitroxyl ions (24). In addition, GSNO is unstable in aqueous solution and undergoes decomposition, which is believed to be homolytic cleavage of the S-N bond to give NO and a thiyl radical (25,26). Indeed, the reactions involving GSNO are fairly complex and generate many potential products including ammonia, NO, nitrous oxide, nitrite, sulfinamide, hydroxylamine, and several oxidized forms of glutathione (20, 23). Recently, it was found that freshly prepared GSNO was effective in S-nitrosylation of proteins through transnitrosylation, whereas the decomposed GSNO was more effective in S-glutathiolation of proteins (10). It was suggested that glutathione sulfenic acid was the active component for glutathiolation of proteins.Neurogranin/RC3 (Ng) and neuromodulin/GAP-43 (Nm) are two prominent PKC substrates in the brain. Phoshorylations of both Ng and Nm reduce their binding affinities for calmodulin (CaM) (27,28). Rat brain Ng contains four, and Nm contains two Cys residues; these Cys residues in Ng form two pairs of intramolecular disulfides upon oxidation by NO and...

Mass Spectrometric Analysis of GAP-43/Neuromodulin Reveals the Presence of a Variety of Fatty Acylated Species

“…With NEMmodified peptides taken into account, ϳ90% of the unacylated Cys residues exist as free sulfhydryl groups in vivo, presumably due to the high concentration of reduced glutathione. The availability of free sulfhydryl groups on cysteines 3 and 4 allows GAP-43 to be additionally modified by ADP-ribosylation (31) and glutathiolation by glutathione disulfide S-oxide (32).…”

Detection, Quantitation, Purification, and Identification of Cardiac Proteins S-Thiolated during Ischemia and Reperfusion

“…It is likely that S-thiolation occurs at the reactive and catalytically essential cysteine 149 (34). Oxidation of this residue inactivates the enzyme and may be achieved by a range of oxidants including GSSG, GSNO, NO, HOCl, and GS(O)SG (33,(35)(36)(37)(38)(39)(40). Triosephosphate isomerase is another glycolytic enzyme that we found to be S-thiolated during cardiac oxidative stress.…”

Ischemia-elicited Oxidative Modulation of Ca2+/Calmodulin-dependent Protein Kinase II

“…Previously, we showed that treatment of rat brain slices with oxidants could generate reactive oxidized glutathione derivatives, GS-DSMO and GS-DSDO, which caused protein glutathionylation and formation of protein disulfide (29). Incubation of the purified CaMKII with GS-DSMO at 0.1 (Fig.…”

“…It is conceivable that these reactive oxygen species could modulate CaMKII activity by direct oxidation of this protein, which constitutes 1-2% of total brain protein (27), as well as by oxidation of its activator, CaM (28). Previously, we showed that activation of NMDA receptors or direct administration of oxidants to the brain slices could trigger thionylation of neurogranin and formation of intra-molecular disulfide of this protein (29,30). The oxidant-induced modification of protein was hypothesized as a result of oxidation by the endogenously produced glutathione disulfide S-oxides (GS-DSOs), which have a high activity to form glutathione-protein-mixed disulfide (glutathionylation) and inter-and/or intra-molecular disulfide bonds of proteins (31)(32)(33).…”