GLUTATHIONE S-TRANSFERASE AND ITS REGULATION OF CARCINOGENIC ELECTROPHILES

谷胱甘肽S-转移酶及其对致癌亲电物质的调控

• 批准号: 6269039
• 负责人: WILLIAM E FAHL
• 金额: $ 22.1万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-06-01 至 1999-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6269039
• 关键词: alkylation; antioxidants; chemical carcinogen; chemical carcinogenesis; chemical conjugate; cytotoxicity; enzyme induction /repression; genetically modified animals; glutathione transferase; human genetic material tag; isozymes; laboratory mouse; membrane transport proteins; molecular oncology; nucleic acid sequence; site directed mutagenesis; toxin metabolism

Glutathione S-transferases have the uncommon characteristic of being able to scavenge and detoxify reactive, electrophilic molecules within the environment of living cells. Experimental results from past work indicate that this ability to conjugate electrophiles to glutathione is essential for the sustained life of a cell. What is now needed to move this research area forward is: i) an understanding of how endogenous GST gene expression is regulated, especially induction, so that chemopreventative molecules can be rationally designed and put into practice, and ii) an understanding of the parameters which determine the extent to which GST/GSH conjunction can be used to capture and detoxify electrophiles in cells, and by so doing, decrease the associated cancer risk. The Aims of our proposed work are designed to bring the concept of preemptive glutathione conjugation of electrophiles to fruition. Our Aims are to: 1) identify how sequence variation in Antioxidant Responsive Elements (AREs) affects inducibility of the dependent, chemoprotective genes, and hence, affects cancer risk, 2) determine the identify of key regulation transcription factor that binds to AREs and regulates the Chemoprotective Response, and 3) develop a comprehensive understanding of the parameters which determine the level of GST/GSH-conferred resistance in mammalian cells. We will use several experimental approaches for these studies, including: analysis of amplified mouse genomic sequences containing AREs, relating ARE DNA sequence to inducibility in an in vitro functional assay as well as inducibility in mouse live; oligonucleotide-based affinity purification of ARE-BP-1, the key ARE-binding transcription factor and cloning of its cDNA; expression of recombinant genes which encode wild-type or mutant, gain-of-function GST isoforms from our current, the GSH biosynthesis enzyme gamma-glutamyl-cysteinesynthase (gammaGCS) or the membrane glutathione-conjugate pump (MRP) in cultured mammalian cells; and the production of two gene-replacement strains of mice, one carrying a knockout of its endogenous GST Yc/1 gene, and one carrying a three codon replacement mutations in the GST Yc/1 gene to confer a gain-of-function phenotype in catalyzing the conjugation of toxic nitrogen mustards. Through these studies, we will work to acheive an understanding of how expression of GST genes is regulated and how endogenous and recombinant GSTs can be used to protect cells from electrophiles.

谷胱甘肽S-转移酶具有不寻常的特性， 去毒和解毒反应性，亲电分子内， 活细胞的环境。过去的实验结果表明， 这种将亲电体与谷胱甘肽结合的能力是必不可少的 for the sustainable持续life of a cell细胞.现在需要什么来推动这项研究 未来的领域是：i）了解内源性GST基因表达 是受调控的，尤其是诱导， 可以合理设计并付诸实践，以及ii）理解 决定GST/GSH结合程度的参数 可用于捕获和解毒细胞中的亲电体， 这样做，降低了相关的癌症风险。我们拟议工作的目标 旨在将抢先谷胱甘肽结合的概念 亲电体结出果实我们的目标是：1）确定序列 抗氧化反应元素（战神）的变化影响诱导 依赖性的化学保护基因，从而影响癌症风险， 2)确定关键调控转录因子的鉴定， 与战神结合并调节化学保护反应，以及3）发展 全面了解决定水平的参数 在哺乳动物细胞中GST/GSH赋予的抗性。 我们将使用几种实验方法进行这些研究，包括： 分析扩增的含有战神的小鼠基因组序列， ARE DNA序列在体外功能测定中也诱导 作为小鼠肝脏中的诱导物;基于阿糖胞苷的亲和纯化 ARE结合关键转录因子ARE-BP-1的克隆及其表达载体的构建 cDNA;编码野生型或突变体的重组基因的表达， 从我们目前的GSH生物合成中获得功能性GST同工型， 酶γ-谷氨酰-半胱氨酸合成酶（γ GCS）或膜 谷胱甘肽共轭泵（MRP）在培养的哺乳动物细胞;和 生产两种基因置换小鼠品系，一种携带一种 敲除其内源性GST Yc/1基因，和一个携带三个密码子 GST Yc/1基因中的置换突变，以赋予功能获得性 表型在催化有毒的氮结合的突变。 通过这些研究，我们将努力了解 GST基因的表达受到调控， GST可用于保护细胞免受亲电体的侵害。