Hydrogen peroxide is a ubiquitous molecule that is formed as a product of many oxidase-mediated reactions and of auto-oxidation of hemoproteins and flavoproteins (38
). As well as being both freely diffusible and reactive itself, H2
must be removed from cells to avoid formation of the highly reactive hydroxyl radical. Detoxification can be mediated by catalases, which catalyze its breakdown into H2
O and O2
, and by glutathione peroxidases, which utilize GSH as a reductant. In yeast, GSH-mediated reactions appear to be the primary means of detoxifying this ROS, although catalases appear to provide an overlapping defense system during particular growth conditions (16
). In addition to the oxidant sensitivity of gsh1
was found to increase the levels of oxidized (GSSG), protein-bound (GSSP), and extracellular GSH at the expense of intracellular GSH (16a
). In the present study, we have shown that the levels of GSSP are increased due to modification of specific target proteins and that Tdh3 is the major S-thiolated protein.
Most of the S-thiolated proteins detected were formed due to reaction with GSH, since this was the most abundant sulfhydryl released by reaction with DTT, and the gsh1
mutant, which is totally devoid of GSH (17
), contained an altered pattern of S-thiolation. Interestingly, the dipeptide γ-Glu-Cys may be able to replace GSH in the S-thiolation reaction, since the pattern observed in the gsh2
mutant was similar to that seen in the wild type. We have previously shown that the gsh2
mutant is devoid of GSH but contains elevated levels of the dipeptide γ-Glu-Gys, which can substitute for GSH as an antioxidant in yeast (18
). The fact that the wild-type pattern of S-thiolation was found in the glr1
mutant, which contains elevated levels of GSSG (15
), argues against a mechanism that proceeds via a thiol-disulfide exchange reaction with the oxidized disulfide form of GSH (GSSG). Similarly, the 2 mM H2
treatment used for these experiments did not cause any increase in the level of GSSG (data not shown).
GAPDH was identified as the most abundant S-thiolated protein in yeast following a challenge with H2
. Previous studies with mammalian cells have also identified GAPDH as a target of S-thiolation, but the studies presented here show for the first time that this is a regulated process and that it affects survival during conditions of oxidative stress. GAPDH is a glycolytic enzyme that is active as a tetramer of identical 37-kDa subunits catalyzing the conversion of glyceraldehyde-3-phosphate to 1,3-diphosphoglycerate. It has been extensively characterized in S. cerevisiae
, in which it is one of the most abundant soluble proteins and is encoded by three unlinked genes designated TDH1
, and TDH3
). McAlister and Holland reported the isolation of yeast mutants lacking combinations of the GAPDH isoenzymes (33
). None of the TDH
genes were individually essential for cell viability, but a functional copy of either TDH2
was required since tdh2 tdh3
double mutants were inviable. These results implied that Tdh1 may perform a function distinct from that of Tdh2 or Tdh3, since Tdh1 alone was unable to support growth. Both the Tdh2 and Tdh3 polypeptides are able to form catalytically active homotetramers, but the apparent Vmax
of the Tdh3 homotetramer is approximately two- to threefold lower than that of the Tdh2 homotetramer (32
). Interestingly, two forms of the Tdh3 polypeptide that differ in their isoelectric charge were detected by two-dimensional PAGE and may represent catalytically different forms of the GAPDH tetramer (32
). The two forms of Tdh3 polypeptide did not arise as a result of differential phosphorylation, and it is tempting to speculate that they arose due to differences in protein S-thiolation. Therefore, the relatively low Vmax
of Tdh3 may be due to the S-thiolated and hence inactivated form of Tdh3.
Given that the Tdh2 and Tdh3 polypeptides have 98% homology and are functionally redundant for glycolysis, it is surprising that they differ in protein S-thiolation. Both Tdh2 and Tdh3 GAPDH enzyme activities were inactivated by exposure to H2O2. However, Tdh3 (thiolated) activity was recovered within 2 h after the removal of the H2O2 stress, suggesting that the oxidative inactivation was reversible. In contrast, Tdh2 (nonthiolated) activity was restored to only 45% of the initial activity during the 2-h recovery period. The S-thiolation of Tdh3 appears to be physiologically relevant since strains lacking TDH3 were hypersensitive to a lethal dose of H2O2 compared to the wild type and tdh2 strains. Thus, S-thiolation, and hence protection of Tdh3 against irreversible oxidation, was required for survival following a challenge with H2O2. However, the nonthiolated Tdh2 polypeptide is also necessary during exposure to oxidative-stress conditions, since it was required for growth during the continued presence of oxidants. Our model to explain these results postulates that during a prolonged exposure to low levels of oxidants, the continued S-thiolation of Tdh3 would negatively affect cell growth, but during such conditions, enough of the Tdh2 enzyme may avoid oxidation to provide the necessary GAPDH activity for growth. At higher concentrations of oxidants, which would irreversibly oxidize the Tdh2 polypeptide, the Tdh3 polypeptide could be protected by S-thiolation until such time as the oxidative pressure is removed (Fig. ).
FIG. 8 Model for the oxidant sensitivity of tdh mutants. During continuous or low exposure to oxidants, Tdh3 is S-thiolated (-SG), inhibiting GAPDH activity. Under these conditions, Tdh2 must provide enough GAPDH activity for growth. Following an exposure to (more ...)
In mammalian cells, the glycolytic synthesis of ATP is inactivated by conditions of oxidative stress, mainly at the level of GAPDH, due to three independent effects (7
): (i) direct inactivation of the enzyme active site, (ii) a decrease in the cofactor NAD, and (iii) a shift in the cytosolic pH from the enzyme optima (7
). Thus, protection of the GAPDH active site by S-thiolation would protect against oxidative inactivation of this crucial glycolytic enzyme. In addition, it has been proposed that blocking glycolysis at the GAPDH step would be beneficial during conditions of oxidative stress since it would result in an increased flux of glucose equivalents through the pentose phosphate pathway, leading to the generation of NADPH (39
). Such NADPH could provide reducing power for antioxidant enzymes including catalases and the GSH-GSH peroxidase system (21
). Evidence is also accumulating that GAPDH functions in processes unrelated to glycolysis, including, DNA repair and replication, translational control of gene expression, and endocytosis and in a plasma membrane oxidoreductase complex (reviewed in references 2
). It is unknown whether S-thiolation of GAPDH affects its activity in these various processes.
In addition to its role in protecting individual protein -SH groups from oxidation, and given the high concentrations (millimolar) of sulfhydryl groups in the cell, thiolation has been suggested to serve as a general antioxidant defense system analogous to free-radical scavengers (47
). However, this seems unlikely from the present data, since the tdh3
mutant, lacking the most abundant S-thiolated protein, did not lead to increases in the level of thiolation of existing proteins or the appearance of new S-thiolated proteins. Rather, the consistent pattern of labelling indicates that S-thiolation is a highly controlled form of posttranslational modification resulting from an oxidant challenge. This modification may serve to protect the cysteine residues of particular proteins from oxidation or, alternatively, may provide a novel form of protein regulation. Two recent reports have indicated a role for reversible thiolation in the regulation of enzyme activity. First, S-thiolation was implicated in the regulation of the HIV-1 protease under the control of glutaredoxin (11
); second, S-thiolation was shown to regulate the E1 and E2 ubiquitin-conjugating enzymes in bovine retina cells (28
). Here, we have demonstrated that the activity of GAPDH in protection against oxidative stress is regulated by protein S-thiolation and that this process is physiologically important for survival during conditions of oxidative stress.