Differential Protein S-Thiolation of Glyceraldehyde-3-Phosphate Dehydrogenase Isoenzymes Influences Sensitivity to Oxidative Stress

Abstract

The irreversible oxidation of cysteine residues can be prevented by protein S-thiolation, in which protein -SH groups form mixed disulfides with low-molecular-weight thiols such as glutathione. We report here the identification of glyceraldehyde-3-phosphate dehydrogenase as the major target of protein S-thiolation following treatment with hydrogen peroxide in the yeast Saccharomyces cerevisiae. Our studies reveal that this process is tightly regulated, since, surprisingly, despite a high degree of sequence homology (98% similarity and 96% identity), the Tdh3 but not the Tdh2 isoenzyme was S-thiolated. The glyceraldehyde-3-phosphate dehydrogenase enzyme activity of both the Tdh2 and Tdh3 isoenzymes was decreased following exposure to H 2 O 2 , but only Tdh3 activity was restored within a 2-h recovery period. This indicates that the inhibition of the S-thiolated Tdh3 polypeptide was readily reversible. Moreover, mutants lacking TDH3 were sensitive to a challenge with a lethal dose of H 2 O 2 , indicating that the S-thiolated Tdh3 polypeptide is required for survival during conditions of oxidative stress. In contrast, a requirement for the nonthiolated Tdh2 polypeptide was found during exposure to continuous low levels of oxidants, conditions where the Tdh3 polypeptide would be S-thiolated and hence inactivated. We propose a model in which both enzymes are required during conditions of oxidative stress but play complementary roles depending on their ability to undergo S-thiolation.Sulfhydryl groups (-SH) play a remarkably broad range of roles in the cell, since the redox status of cysteine residues is involved in both the structure and function of numerous enzymes, receptors, and transcription factors. However, cysteine residues are among the most easily oxidized residues in proteins, and the oxidation of -SH groups is one of the earliest observable events during reactive oxygen species (ROS)-mediated damage (6, 12). The oxidation of -SH groups can result in the formation of protein disulfides (S-S) through two protein cysteines or mixed disulfides between protein-SH groups and a number of low-molecular-weight thiols (protein S-thiolation). While the role of disulfide bond formation in protein folding has been well characterized (see, for example, reference 23), its role during conditions of oxidative stress is unclear. In contrast, protein S-thiolation has been proposed to serve an antioxidant function by preventing the irreversible oxidation of cysteine residues to higher oxidation states (e.g., sulfenic acid) following exposure to ROS (10, 47).Modification of proteins by S-thiolation does not require an enzymatic activity and has been proposed to occur either by the reaction of partially oxidized protein sulfhydryls (thiyl radical or sulfenic acid intermediates) with thiols such as cysteine or glutathione (GSH) or by thiol-disulfide exchange reactions with the oxidized disulfide form of GSH (GSSG) (47). To provide a defense against conditions of oxidative stress, this modification must be reversible. Dethiolatio...

Regulatory crosstalk of the metabolic network

“…A few minutes later, a genetic response is induced, by which the PPP is upregulated and glycolytic enzymes are downregulated [70]. After the transcriptional reconfiguration of the pathway is complete ($30-60 minutes), the GAPDH block is released and the metabolic flux is re-established [71,72]. The transcriptional response occurs in discrete temporal steps; in yeast, upregulation of the first mRNA targets is detected 3-5 minutes after stress induction.…”

Cellular Defenses against Superoxide and Hydrogen Peroxide

“…Glyceraldehyde-3-phosphate dehydrogenase, for example, has received attention because it is inactivated when eukaryotic cells are stressed by moderate doses (ca. 50 μM) of exogenous H 2 O 2 (108). The enzyme employs a cysteine thiolate as a nucleophile; a histidine residue opposes the substate binding site, where in the course of normal catalysis it stabilizes and then protonates the reaction intermediate (109).…”

The Pep4p vacuolar proteinase contributes to the turnover of oxidized proteins but PEP4 overexpression is not sufficient to increase chronological lifespan in Saccharomyces cerevisiae

“…Protein oxidative modifications include the reversible oxidation of sulphurcontaining amino acids, and the irreversible conversion of some amino acid residues to carbonyl derivatives. The oxidation of protein cysteine residues generates sulphenic acid derivatives that are S-thiolated with reduced glutathione (Grant et al, 1999;Demasi et al, 2003;Shenton & Grant, 2003), or further oxidized to sulphinic acid moieties. Protein activity is restored by the monothiol glutaredoxin Grx5p (Shenton et al, 2002) or by sulphiredoxin (Biteau et al, 2003), respectively.…”