REDOX REGULATION OF TRANSCRIPTION FACTOR FUNCTION

活性氧(ROS)是正常细胞代谢的副产物。这些ROS的不适当水平与许多重要的人类健康问题的病理生理学有关，从癌症到衰老的影响。我们正在使用酿酒酵母作为真核细胞解毒ROS能力的模型系统。我们实验室和其他实验室的工作已经证明，酿酒酵母YAP-1转录调控蛋白对这种微生物在氧化剂暴露下生存至关重要。哺乳动物的c-jun转录是YAP-1的同源基因，已被证明参与动物细胞对氧化应激的反应，这表明YAP-1和c-jun的这一作用可能在进化上是保守的。我们的初步实验表明，YAP-1的突变形式根据用来挑战细胞的氧化剂类型的不同而表现不同。YAP-1的截短突变形式对联胺胁迫高度抵抗，但对过氧化氢高度敏感。我们已经证明，氧化应激对YAP-1的控制发生在翻译后步骤，需要在因子的C末端有半胱氨酸残基。为了探索这些半胱氨酸残基作为潜在的氧化还原传感器的作用，我们将使用半胱氨酸修饰的化学探针来评估它们的反应性。肽图谱将被用来检测蛋白质的翻译后修饰。氧化剂对野生型和突变型Yap1p亚细胞定位的影响将通过与转录因子的绿色荧光蛋白融合来检测。我们推测，截短形式的YAP-1在提供比正常情况下更强的联胺耐受性的同时，未能赋予H_2O_2抗性是由于这些突变因子在面对H_2O_2挑战时激活靶基因表达的缺陷。支持这一观点的是，依赖Yap1p的过氧化氢抗性基因TRX2通常不受Yap1p突变形式的调控。建议通过实验来确定TRX2启动子对氧化剂和Yap1p的特征反应所需的特征。为了确定在过氧化氢抗性基因中影响YAP-1功能的蛋白质，将分离允许YAP-1的截断突变形式赋予过氧化氢抗性的第二位点抑制物。这组实验的成功完成将描绘导致氧化应激激活YAP-1的事件，并为蛋白质功能的氧化还原控制背后的机制提供新的见解。

Reactive oxygen species (ROS) are a by-product of a normal cellular metabolism. Inappropriate levels of these ROS has been implicated in the pathophysiology of a number of important human health concerns, ranging from cancer to the effects of aging. We are employing the yeast Saccharomyces cerevisiae as a model system for the ability of eukaryotic cells to detoxify ROS. Work from our laboratory and others has demonstrated that the S. cerevisiae yAP-1 transcriptional regulatory protein is crucial for this organism to survive oxidant exposure. The mammalian c- Jun transcription, a yAP-1 homolog, has been shown to be involved in the response of animal cells to oxidative stress, suggesting the possible evolutionary conservation of this role of yAP-1 and c-Jun. Our preliminary experiments have demonstrated that mutant forms of yAP-1 behave differently depending on the type of oxidant used to challenge the cells. Truncated mutant forms of yAP-1 are hyper-resistant to diamide stress but hypersensitive to H2O2. We have demonstrated that control of yAP-1 by oxidative stress occurs at a posttranslational step that requires cysteine residues in the C-terminus of the factor. To explore role of these cysteine residues as potential redox sensors, we will evaluate their reactivity using a chemical probe for cysteine modification. Peptide mapping will be used to detect post- translational modifications of the protein. The influence of oxidants on subcellular localization of wild-type and mutant forms of Yap1p will be examined using green fluorescent protein fusions to the transcription factor. We hypothesize that the failure of truncated forms of yAP-1 to confer H2O2 resistance while providing greater-than-normal diamide tolerance is due to a defect in these mutant factors to activate target gene expression in the face of H2O2 challenge. In support of this idea, the Yap1p-dependent H2O2 resistance gene TRX2 is not normally regulated by mutant forms of Yap1p. Experiments are proposed to determine the features of the TRX2 promoter that are required to confer its characteristic response to oxidants and Yap1p. To identify proteins that act to influence the function of yAP-1 at H2O2 resistance genes, second- site suppressors will be isolated that allow the truncated mutant forms of yAP-1 to confer H2O2 resistance. The successful completion of this set of experiments will delineate the events that lead to activation of yAP-1 by oxidative stress and provide new insight into the mechanisms behind redox control of protein function.