Centro de Química e Bioquímica, Faculdade de Ciências, Universidade de Lisboa, Campo Grande, 1749-016 Lisboa, Portugal.
BMC Biochem. 2010 Dec 28;11:49. doi: 10.1186/1471-2091-11-49.
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.
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.
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.
蛋白质 SH 基团的可逆氧化被认为是氧化还原调节的基础，通过这种调节，过氧化氢（H2O2）浓度的变化可以控制蛋白质的功能。在许多细胞系统中，过氧化氢暴露后，几种蛋白质会发生 S-谷胱甘肽化。在酵母中，当使用初始高剂量的 H2O2 时，甘油醛-3-磷酸脱氢酶（GAPDH）被确定为 S-谷胱甘肽化的主要靶标，导致酶的可逆失活。H2O2 对 GAPDH 的失活作用是将碳水化合物通量重新路由以产生 NADPH。在这里，我们报告了低调节 H2O2 剂量对酿酒酵母中 GAPDH 活性和表达的影响。
本研究采用了一种校准和控制 H2O2 输送的方法 - 稳态滴定法 - 其中细胞暴露于恒定、低且已知的 H2O2 浓度下。与常见的一次性添加相反，这种技术允许确定哪些 H2O2 浓度引发特定的生物学反应。这项工作表明，在指数期和静止期细胞中，低调节 H2O2 浓度诱导过氧化氢酶的大量上调，这是细胞氧化应激反应的特征指纹，但只有在诱导死亡的高 H2O2 剂量下才观察到 GAPDH 氧化和随之而来的活性下降。GAPDH 活性在孵育亚致死 H2O2 剂量时保持不变，但在静止期细胞中，三种 GAPDH 同工酶的表达存在差异反应：Tdh1p 被强烈上调，而 Tdh2p/Tdh3p 则略有下调。
在酵母中，GAPDH 活性对低至中等 H2O2 剂量基本无反应。这表明了一种情况，即（a）细胞内氧化还原酶能够有效地应对由广泛的亚致死 H2O2 浓度诱导的 GAPDH 氧化水平，（b）GAPDH 的失活不能被认为是体内 H2O2 诱导氧化的敏感生物标志物。由于 GAPDH 失活仅发生在诱导细胞死亡的高 H2O2 剂量下，因此 GAPDH 依赖性碳水化合物通量重路由可能仅在病理生理情况下很重要。这项工作强调了使用更接近生理浓度研究 H2O2 诱导的氧化应激对于确定蛋白质氧化现象在细胞代谢调节中的重要性的重要性。
