GLUTATHIONE S-TRANSFERASE AND ITS REGULATION OF CARCINOGENIC ELECTROPHILES

谷胱甘肽S转移酶具有不寻常的特征，即能够 清除和解毒体内活性的亲电分子 活细胞的环境。过去工作的实验结果表明 这种将亲电体与谷胱甘肽偶联的能力是必不可少的 为了细胞的持续寿命。现在需要什么来推动这项研究 前沿领域是：i)对内源性GST基因表达的理解 是受调控的，尤其是诱导，所以化学预防分子 能够合理设计并付诸实施，以及二)理解 哪些参数决定了GST/GSH结合的程度 可以用来捕获细胞中的亲电体并对其进行解毒，因此 这样做，可以降低相关的癌症风险。我们拟议工作的目标 旨在带来先发制人谷胱甘肽结合的概念 从亲电到结实。我们的目标是：1)确定序列 抗氧化剂反应元件(ARs)的变化影响诱导性 依赖的，化学保护基因，因此，影响癌症风险， 2)确定关键调控转录因子的鉴定 与ARES结合并调节化学保护反应，3)发展 全面了解决定水平的参数 在哺乳动物细胞中，GST/GSH-赋予抗性。 我们将使用几种实验方法进行这些研究，包括： 含ARES基因的小鼠基因组扩增序列分析 DNA序列在体外功能测定中是否也具有诱导性 AS在小鼠体内的诱导性；基于寡核苷酸的亲和纯化 ARE-BP-1关键的ARE结合转录因子及其克隆 编码野生型或突变型重组基因的表达， 从我们目前的GSH生物合成中获得的功能增益GST亚型 谷氨酰半胱氨酸合酶(GammaGCS)或膜 培养的哺乳动物细胞中的谷胱甘肽共轭泵(MRP)； 产生两个基因替换小鼠品系，其中一个携带A基因 其内源性GST YC/1基因被敲除，其中一例携带三个密码子 GST YC/1基因的替换突变导致功能获得 有毒氮素芥菜催化接合的表型。 通过这些研究，我们将努力了解如何 GST基因的表达调控及其内源性和重组性 GSTS可以用来保护细胞免受亲电性物质的伤害。

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.