| Abstract |
Background: The reversible oxidation of protein SH groups has been considered to be the basis of redox regulation by which changes in hydrogen peroxide (H2O2) concentrations may control protein function. Several proteins become S-glutathionylated following exposure to H2O2 in a variety of cellular systems. In yeast, when using a high initial H2O2 dose, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified as the major target of S-glutathionylation which leads to reversible inactivation of the enzyme. GAPDH inactivation by H2O2 functions to reroute carbohydrate flux to produce NADPH. Here we report the effect of low regulatory H2O2 doses on GAPDH activity and expression in Saccharomyces cerevisiae. Results: A calibrated and controlled method of H2O2 delivery - the steady-state titration - in which cells are exposed to constant, low, and known H2O2 concentrations, was used in this study. This technique, contrary to the common bolus addition, allows determining which H2O2 concentrations trigger specific biological responses. This work shows that both in exponential-and stationary-phase cells, low regulatory H2O2 concentrations induce a large upregulation of catalase, a fingerprint of the cellular oxidative stress response, but GAPDH oxidation and the ensuing activity decrease are only observed at death-inducing high H2O2 doses. GAPDH activity is constant upon incubation with sub-lethal H2O2 doses, but in stationary-phase cells there is a differential response in the expression of the three GAPDH isoenzymes: Tdh1p is strongly upregulated while Tdh2p/Tdh3p are slightly downregulated. Conclusions: In yeast GAPDH activity is largely unresponsive to low to moderate H2O2 doses. This points to a scenario where (a) cellular redoxins efficiently cope with levels of GAPDH oxidation induced by a vast range of sub-lethal H2O2 concentrations, (b) inactivation of GAPDH cannot be considered a sensitive biomarker of H2O2-induced oxidation in vivo. Since GAPDH inactivation only occurs at cell death-inducing high H2O2 doses, GAPDH-dependent rerouting of carbohydrate flux is probably important merely in pathophysiological situations. This work highlights the importance of studying H2O2-induced oxidative stress using concentrations closer to the physiological for determining the importance of protein oxidation phenomena in the regulation of cellular metabolism. |
