NOVEL GST AND DETOXICATION OF DIOL EPOXIDES

新型 GST 和二醇环氧化物的解毒

• 批准号: 6344500
• 负责人: Shivendra Singh
• 金额: $ 23.12万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-01 至 2002-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6344500
• 关键词: adduct; affinity chromatography; animal tissue; benzopyrenediol epoxide; carcinogenesis inhibitor; chemoprevention; detoxification; enzyme activity; enzyme structure; glutathione transferase; high performance liquid chromatography; human tissue; isozymes; laboratory mouse; liver cells; molecular cloning; plant extracts; recombinant proteins; site directed mutagenesis; species difference

The overall objective of this project is to define the role of a functionally unique alpha class murine glutathione (GSH) transferase (GST) (designated as ~mouse GST 9.5") and its human orthologue(s) in cellular protection against carcinogenic diol epoxides (Des) of polycyclic aromatic hydrocarbons, which are widespread environmental pollutants. Recent studies from our laboratory indicate that GST 9.5 is a heterodimer of A1 and A2 type murine GST subunits. The hypothesis of this project is that mouse GST 9.5 (mGSTA1-2), which, unlike other alpha class mammalian GSTs, is exceptionally active toward Des, and its human orthologue(s) may play an important role in the detoxication of Des. This hypothesis will be tested by: (I) determining the kinetic constants for tissue-isolated murine GSTs, including GST 9.5, as well as the recombinant mGSTA1-1 and m GSTA2-2 homodimers, which were recently produced by us, in the GSH conjugation of various bay-and fjord-region Des, (ii) identifying the critical amino acids(s) which may account for the relatively higher catalytic efficiency of the A1 type subunit of GST 9.5 toward Des as compared with the A2 type subunit (through site-directed mutagenesis and kinetic characterization of the mutants), (iii) determining the expression of GST 9.5 subunits in other A/J mouse tissues by GSH- affinity chromatography followed by reverse-phase HPLC, (iv) determine the effects of expression of GST 9.5 subunits in Hepa-1 cells, through stable transfection, on cytotoxicity of (+)-anti-BPDE (trans-N2dG-BPD adduct), (v) determining the correlation in vivo between chemopreventive efficacies of six anti-carcinogenic organosulfides from garlic against BP-induced cancer of forestomach and lung in A/J mice wit their effects on the levels of various GSTs, including GST 9.5, in the liver and target organs, (vi) purifying from stomach and/or liver of the human orthologue(s) of murine GST 9.5 for its/their structural, immunological and kinetic characterization, and (vii) determining the expression of human orthologue(s) of GST 9.5 in other human tissues. These studies will not only enhance our understanding of the protective mechanisms against carcinogenic Des but also shed light on the role of mouse GST 9.5 and its orthologue(s) in the detoxication of these carcinogens. In the long term, strategies to manipulate this group of GSTs to the advantage of cells/organ should be extremely important for cancer prevention.

该项目的总体目标是确定一个 功能独特的α类鼠谷胱甘肽（GSH）转移酶 (GST)（指定为~小鼠GST 9.5”）及其人直向同源物， 细胞对致癌的二醇环氧化物（Des）的保护 多环芳烃，这是广泛的环境 污染物 我们实验室最近的研究表明，GST 9.5 是A1和A2型鼠GST亚基的异源二聚体。 的 本项目假设是小鼠GST 9.5（mGSTA 1 -2）， 与其他α类哺乳动物GST不同， Des及其人类直系同源物可能在以下方面发挥重要作用： Des的解毒作用 这一假设将通过以下方式进行检验：（一） 确定组织分离的鼠GST的动力学常数， 包括GST 9.5，以及重组mGSTA 1 -1和mGSTA 2 -2 同二聚体，这是我们最近生产的，在谷胱甘肽 共轭的各种海湾和峡湾地区Des，（ii）确定 关键氨基酸，可能导致相对较高的 GST 9.5的A1型亚基对Des的催化效率为 与A2型亚基相比（通过定点突变 和突变体的动力学表征），（iii）确定突变体的 谷胱甘肽转移酶9.5亚基在其他A/J小鼠组织中的表达 亲和色谱法，随后进行反相HPLC，（iv） 探讨GST 9.5亚基在Hepa-1细胞中表达的影响 细胞，通过稳定转染，对（+）-抗BPDE的细胞毒性 （v）确定体内相关性 六种抗癌药物的化学预防效果 大蒜中有机硫化物对BP诱发前胃癌的抑制作用 和肺组织中各种GST水平的影响， 包括GST 9.5，在肝脏和靶器官中，（vi）纯化 来自鼠GST的人直系同源物的胃和/或肝 9.5其结构、免疫学和动力学 表征，和（vii）确定人的表达， GST 9.5在其他人体组织中的直向同源物。 这些研究将 不仅加深了我们对保护机制的理解 对抗致癌的Des，也揭示了小鼠GST的作用 9.5及其直向同源物在这些致癌物的解毒中的作用。 从长远来看，操纵这群GST的策略， 细胞/器官的优势对癌症应该是非常重要的 预防