PROTEIN THIOLS AND MECHANISMS OF TOXICITY

本项目的主要目标是阐明 烷基化对蛋白质的结构和生物学功能的影响， 含有“硫氧还蛋白折叠”。与谷胱甘肽（GSH）结合后 1，2-二卤乙烷、1，2-二氯乙烷（DCE）和1，2-二溴乙烷 (DBE，通过各自的表锍离子变成烷基化剂， S-（2-氯乙基）谷胱甘肽（CEG）和S-（2-溴乙基）谷胱甘肽（BEG）。 这些偶联物已显示在约50-100 ℃下烷基化蛋白质 乘以核酸烷基化的速率。CEG的体外研究 显示重组大肠杆菌硫氧还蛋白的Cys 32位置是 与任何其他氨基酸残基相比优先烷基化，而 血红蛋白的烷基化要低一个数量级 在相同条件下进行烷基化。在本提案中，我们假设， 硫氧还蛋白折叠，Cys-X-Y-Cys，存在于几个 “硫氧还蛋白折叠”家族的蛋白质，包括硫氧还蛋白（TRX）， 硫氧还蛋白还原酶（TR）和蛋白质二硫键异构酶（PDI）是 DCE和DBE的谷胱甘肽缀合物的烷基化靶点， 蛋白质的生物学功能在 在其活性部位进行烷基化。PDI高度参与蛋白质 在内质网中蛋白质合成期间折叠。位点 将建立重组哺乳动物TRX、TR和PDI的烷基化 正如本实验室用E.大肠杆菌硫氧还蛋白。烷基化 除CEG外的试剂包括1-氯-2，4-二硝基苯（DNCB）和 碘乙酰胺重组人巨噬细胞集落刺激因子β （rhm-CSF β），一种细胞因子，被用作模型蛋白， 蛋白质折叠的研究，将进行调查，以确定影响 CEG烷基化对蛋白质折叠和生物活性的影响。影响 蛋白质折叠、转录因子活化和氧化还原 将进行修改。

The main objective of this project is to elucidate the effects of alkylation on the structure and biological functions of proteins that contain the "thioredoxin fold". Upon conjugation with glutathione (GSH) the 1,2-dihaloethanes, 1,2-dichloroethane (DCE) and 1,2-dibromoethane (DBE, become alkylating agents via the episulfonium ion of the respective S-(2-chloroethyl) glutathione (CEG) and S-(2-bromoethyl)glutathione (BEG). These conjugates have been shown to alkylate proteins at about 50-100 times the rate of nucleic acid alkylation. In vitro studies with CEG have shown that the Cys32 position of recombinant E.coli thioredoxin is preferentially alkylated compared to any other amino acid residue, whereas the alkylation of hemoglobulin is over one order of magnitude less alkylated under the same conditions. In this proposal, we hypothesize that the thioredoxin fold, Cys-X-Y-Cys, present in the active site of several proteins of the "thioredoxin fold" family, including thioredoxin (TRX), thioredoxin reductase (TR), and protein disulfide isomerase (PDI) are targets for alkylation by the glutathione conjugates of DCE and DBE and that the biological functions of the proteins are highly compromised after alkylation at their active sites. PDI is highly involved in protein folding during protein synthesis in the endoplasmic reticulum. Sites of alkylation will be established for recombinant mammalian TRX, TR and PDI as this laboratory has accomplished with E. coli thioredoxin. Alkylating agents besides CEG include 1-chloro-2,4-dinitrobenzene (DNCB) and iodoacetamide. Recombinant human macrophage-colony stimulating factor beta (rhm-CSFbeta), a cytokine that is being used as a model protein for protein folding studies, will be investigated to determine the effects of CEG alkylation on protein folding and biological activity. Effects on protein folding, activation on transcription factors, and redox modifications will be investigated.