Protein S-thiolation targets glycolysis and protein synthesis in response to oxidative stress in the yeast Saccharomyces cerevisiae

Abstract

The irreversible oxidation of cysteine residues can be prevented by protein S-thiolation, a process by which protein SH groups form mixed disulphides with low-molecular-mass thiols such as glutathione. We report here the target proteins which are modified in yeast cells in response to H(2)O(2). In particular, a range of glycolytic and related enzymes (Tdh3, Eno2, Adh1, Tpi1, Ald6 and Fba1), as well as translation factors (Tef2, Tef5, Nip1 and Rps5) are identified. The oxidative stress conditions used to induce S-thiolation are shown to inhibit GAPDH (glyceraldehyde-3-phosphate dehydrogenase), enolase and alcohol dehydrogenase activities, whereas they have no effect on aldolase, triose phosphate isomerase or aldehyde dehydrogenase activities. The inhibition of GAPDH, enolase and alcohol dehydrogenase is readily reversible once the oxidant is removed. In addition, we show that peroxide stress has little or no effect on glucose-6-phosphate dehydrogenase or 6-phosphogluconate dehydrogenase, the enzymes that catalyse NADPH production via the pentose phosphate pathway. Thus the inhibition of glycolytic flux is proposed to result in glucose equivalents entering the pentose phosphate pathway for the generation of NADPH. Radiolabelling is used to confirm that peroxide stress results in a rapid and reversible inhibition of protein synthesis. Furthermore, we show that glycolytic enzyme activities and protein synthesis are irreversibly inhibited in a mutant that lacks glutathione, and hence cannot modify proteins by S-thiolation. In summary, protein S-thiolation appears to serve an adaptive function during exposure to an oxidative stress by reprogramming metabolism and protecting protein synthesis against irreversible oxidation.

Changes in disulfide bond content of proteins in a yeast strain lacking major sources of NADPH

“…In the latter mutant, Tpi1p and Adh1p were highly oxidized, consistent with our observations of disulfide bonds in isoforms of these enzymes in the Δidp2Δzwf1 mutant strain. There are also some interesting correlations between results of these proteomic screens for cysteine oxidation [55, current study] with those obtained by Shenton and Grant [17] who examined yeast proteins that were S-thiolated, i.e., that formed mixed disulfide bonds with compounds like glutathione, in response to an acute challenge with hydrogen peroxide. Commonly labeled proteins included the glycolytic enzymes Tpi1p, Tdh3p, Eno2p, and Adh1p.…”

“…A protein translation factor, Tef2p, which demonstrated disulfide bond content in some of our cellular extracts, was also identified as an S-thiolated protein. Shenton and Grant [17] found that S-thiolation correlated with a reduction in activities of several glycolytic enzymes and in rates of protein synthesis. These effects could be reversed by removal of hydrogen peroxide, suggesting that S-thiolation functions to redirect metabolic flux through the pentose phosphate pathway to increase production of NADPH during transient exogenous oxidative challenge.…”

“…However, it is also possible that some of the cysteine side chains have been oxidized to forms that would not be detected in our screen. What is quite clear in a comparison of the current and other global analyses of yeast proteins [17,55] is that there are only a limited number of cellular proteins that contain reactive cysteine side chains, and most of these proteins are cytosolic. Also, the oxidative status of the cysteine residues in these proteins appears to be a good index for cellular redox status.…”

“…This form of cysteine oxidation would encompass intramolecular or intermolecular disulfide bonds. The latter would include S-thiolation, the formation of a disulfide bond between a protein and a low molecular weight cofactor like glutathione [16,17]. It has been suggested that this type of disulfide bond formation may protect proteins during transient oxidative stress until levels of reactive oxygen species decline and reducing conditions are restored [17].…”

“…The latter would include S-thiolation, the formation of a disulfide bond between a protein and a low molecular weight cofactor like glutathione [16,17]. It has been suggested that this type of disulfide bond formation may protect proteins during transient oxidative stress until levels of reactive oxygen species decline and reducing conditions are restored [17]. Cysteine side chains can also undergo oxidation to sulfinic or sulfonic acids, oxidative products that are not reduced by normal cellular thiol-based antioxidant systems [16,18] and that would not be detected in our disulfide bond screen.…”