REDOX REGULATION OF TRANSCRIPTION FACTOR FUNCTION

转录因子功能的氧化还原调节

• 批准号: 6525429
• 负责人: W Scott Moye-Rowley
• 金额: $ 15.71万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-01 至 2004-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6525429
• 关键词: cysteine; gene mutation; hydrogen peroxide; oxidative stress; posttranslational modifications; protein localization; protein structure function; site directed mutagenesis; transcription factor

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.

活性氧（ROS）是正常细胞代谢的副产物。 这些ROS的不适当水平已经涉及许多重要的人类健康问题的病理生理学，从癌症到衰老的影响。 我们使用酵母酿酒酵母作为真核细胞解毒活性氧能力的模型系统。 我们实验室和其他实验室的工作表明，S。酿酒酵母雅普-1转录调节蛋白是该生物体在氧化剂暴露下存活的关键。 哺乳动物c-Jun转录，雅普-1同源物，已被证明参与动物细胞对氧化应激的反应，这表明雅普-1和c-Jun的这种作用可能是进化保守的。 雅普-1的截短突变体形式对二酰胺胁迫具有超抗性，但对H2 O2超敏感。 我们已经证明，控制雅普-1的氧化应激发生在翻译后的步骤，需要在C-末端的因子的半胱氨酸残基。 为了探索这些半胱氨酸残基作为潜在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。 肽图谱将用于检测蛋白质的翻译后修饰。 将使用绿色荧光蛋白融合到转录因子中，研究氧化剂对野生型和突变型Yap 1 p亚细胞定位的影响。我们推测，截短形式的雅普-1赋予H2 O2抗性，同时提供比正常的二酰胺耐受性的失败是由于这些突变因子在面对H2 O2挑战时激活靶基因表达的缺陷。支持这一观点的是，Yap 1 p依赖的H2 O2抗性基因TRX 2通常不受Yap 1 p突变形式的调控。实验提出，以确定TRX 2启动子，需要赋予其特征性的响应氧化剂和Yap 1 p的功能。 为了鉴定影响雅普-1在H2 O2抗性基因中的功能的蛋白质，将分离第二位点抑制子，其允许雅普-1的截短突变形式赋予H2 O2抗性. 这组实验的成功完成将描绘的事件，导致激活的雅普-1的氧化应激和提供新的见解背后的氧化还原控制蛋白质功能的机制。