Toxicant-Induced Nuclear Translocation of Thioredoxin

有毒物质诱导的硫氧还蛋白核易位

• 批准号: 6614166
• 负责人: WALTER H WATSON
• 金额: $ 10.75万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-08 至 2006-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6614166
• 关键词: HeLa cells; SDS polyacrylamide gel electrophoresis; active sites; biological signal transduction; cell nucleus; confocal scanning microscopy; environmental exposure; intracellular transport; nuclear factor kappa beta; oxidation; oxidation reduction reaction; oxidative stress; polymerase chain reaction; protein localization; protein transport; site directed mutagenesis; thioredoxin; tissue /cell culture; transcription factor; western blottings

DESCRIPTION (provided by applicant) Thioredoxin (Trx) is a redox-active protein that plays a fundamental role in the cellular responses to oxidants, heavy metals, and alkylating agents. The Trx system scavenges peroxides, repairs damaged proteins, provides bases for the DNA repair machinery, and facilitates the expression of protective genes through the reduction of key transcription factors. Trx is mainly localized to the cytoplasm, but translocates to the nucleus in response to toxic and inflammatory stimuli. However, the factors regulating this redistribution are unknown. In addition to the two active site cysteines (Cys), Trx contains three other Cys residues, the function of which is unknown. This proposal seeks to test the hypothesis that oxidation of Trx signals its redistribution to the nucleus. This will be possible due to the recent development of the Redox Western blot technique, which measures the redox state of the active site and structural Cys residues in Trx. The first aim of this proposal is to test the hypothesis that oxidants in general induce the translocation of Trx to the nucleus, and that this is associated with an oxidation of Trx. The second aim is to determine whether oxidation of Trx is an event that is common to known inducers of Trx translocation (cytokines, phorbol esters, ultraviolet light and ionizing radiation, cobalt chloride, and hypoxia). The third aim is to identify the amino acid residues that are necessary for translocation of Trx to the nucleus. To accomplish this, each of the five Cys residues will be individually mutated to Serines via site-directed mutagenesis, and the subcellular localization of the mutant Trx will be assessed before and after exposure to oxidants or other inducers of nuclear translocation. Successful completion of these aims will define the redox state of nuclear and cytoplasmic Trx in cells exposed to toxic and inflammatory stimuli, and will show whether thiol oxidation contributes to the dynamic control of Trx Iocalization within cells. This information will provide the basis for future investigations into the role of nuclear Trx in redox-dependent processes such as transcriptional up-regulation of protective genes.

描述（由申请人提供） 硫氧还蛋白（Trx）是一种氧化还原活性蛋白，在细胞对氧化剂、重金属和烷化剂的反应中起重要作用。 Trx系统清除过氧化物，修复受损蛋白，为DNA修复机制提供基础，并通过减少关键转录因子促进保护性基因的表达。Trx主要定位于细胞质，但响应于毒性和炎症刺激而移位至细胞核。 然而，调节这种再分配的因素尚不清楚。 除了两个活性位点半胱氨酸（Cys）外，Trx还含有三个其他Cys残基，其功能尚不清楚。 该提案旨在检验Trx氧化信号重新分配到细胞核的假设。 这将是可能的，由于最近开发的氧化还原蛋白质印迹技术，它测量的氧化还原状态的活性位点和结构的半胱氨酸残基的Trx。 该建议的第一个目的是检验氧化剂通常诱导Trx易位到细胞核的假设，并且这与Trx的氧化有关。 第二个目的是确定Trx的氧化是否是已知的Trx易位诱导剂（细胞因子、佛波醇酯、紫外线和电离辐射、氯化钴和缺氧）常见的事件。 第三个目的是鉴定Trx转运到细胞核所必需的氨基酸残基。 为了实现这一点，五个Cys残基中的每一个将通过定点诱变单独突变为丝氨酸，并且在暴露于氧化剂或其他核易位诱导剂之前和之后评估突变体Trx的亚细胞定位。 这些目标的成功完成将定义暴露于毒性和炎症刺激的细胞中细胞核和细胞质Trx的氧化还原状态，并将显示硫醇氧化是否有助于细胞内Trx定位的动态控制。 这些信息将为今后研究核Trx在氧化还原依赖过程中的作用提供基础，如保护基因的转录上调。