REDOX REGULATION OF TRANSCRIPTION FACTOR FUNCTION

转录因子功能的氧化还原调节

• 批准号: 6045185
• 负责人: W Scott Moye-Rowley
• 金额: $ 15.89万
• 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/6045185
• 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品。 这些活性氧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。 我们使用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，酿酒酵母 yAP-1 转录调节蛋白对于该生物体在氧化剂暴露下生存至关重要。 哺乳动物 c-Jun 转录是 yAP-1 同源物，已被证明参与动物细胞对氧化应激的反应，表明 yAP-1 和 c-Jun 的这种作用可能在进化中保守。我们的初步实验表明，yAP-1 的突变形式根据用于攻击细胞的氧化剂类型而表现不同。 yAP-1 的截短突变体对二酰胺胁迫具有超抗性，但对 H2O2 超敏感。 我们已经证明，氧化应激对 yAP-1 的控制发生在翻译后步骤，需要该因子 C 末端的半胱氨酸残基。 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。 将使用与转录因子融合的绿色荧光蛋白来检查氧化剂对 Yap1p 野生型和突变型亚细胞定位的影响。我们假设，截短形式的 yAP-1 未能赋予 H2O2 抗性，同时提供高于正常的二酰胺耐受性，是由于这些突变因子在面临 H2O2 挑战时激活靶基因表达的缺陷。为了支持这一观点，Yap1p 依赖性 H2O2 抗性基因 TRX2 通常不受 Yap1p 突变形式的调节。建议通过实验来确定 TRX2 启动子的特征，这些特征是赋予其对氧化剂和 Yap1p 特征反应所需的。 为了鉴定影响 yAP-1 在 H2O2 抗性基因上的功能的蛋白质，将分离第二位点抑制子，其允许 yAP-1 的截短突变形式赋予 H2O2 抗性。 这组实验的成功完成将描述氧化应激导致 yAP-1 激活的事件，并为氧化还原控制蛋白质功能背后的机制提供新的见解。