Toxicant-Induced Nuclear Translocation of Thioredoxin

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发挥重要作用。TRX系统清除过氧化物质，修复受损的蛋白质，为DNA修复机制提供基础，并通过减少关键转录因子来促进保护性基因的表达。Trx主要定位于细胞质，但在毒性和炎症刺激下转位到细胞核。然而，调控这种再分配的因素尚不清楚。除了两个活性部位半胱氨酸(Cys)外，Trx还含有另外三个Cys残基，其功能尚不清楚。这一提议试图检验这样一种假设，即Trx的氧化发出信号，将其重新分配到原子核。这将是可能的，这要归功于最近氧化还原Western印迹技术的发展，该技术测量了TRX中活性部位和结构半胱氨酸残基的氧化还原状态。这一提议的第一个目的是检验这样的假设，即氧化剂一般诱导Trx移位到核，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易位诱导者(细胞因子、佛波酯、紫外线和电离辐射、氯化钴和低氧)共同的事件。第三个目的是确定Trx转位到细胞核所必需的氨基酸残基。为了做到这一点，五个半胱氨酸残基将通过定点突变单独突变为丝氨酸，突变的Trx的亚细胞定位将在暴露于氧化剂或其他核易位诱导剂之前和之后进行评估。这些目标的成功完成将确定暴露在有毒和炎症刺激下的细胞中核和细胞质Trx的氧化还原状态，并将表明硫醇氧化是否有助于Trx在细胞内定位的动态控制。这些信息将为进一步研究核Trx在氧化还原依赖过程中的作用提供基础，例如保护基因的转录上调。

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.