Novel GST and Detoxication of Diol Epoxides

新型 GST 和二醇环氧化物的解毒

• 批准号: 6606766
• 负责人: Shivendra Singh
• 金额: $ 28.05万
• 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 2007-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6606766
• 关键词: SDS polyacrylamide gel electrophoresis; X ray crystallography; active sites; affinity chromatography; carbopolycyclic compound; carcinogens; detoxification; diol; environment; enzyme activity; enzyme substrate; epoxides; glutathione transferase; hazardous substances; high performance liquid chromatography; human tissue; isozymes; liver; lung; postmortem; site directed mutagenesis; western blottings

DESCRIPTION (provided by applicant): During the current funded period of this grant, we investigated the role of murine glutathione (GSH) transferases (GSTs) in detoxification of activated metabolites (diol epoxides) of polycyclic aromatic hydrocarbons (PAHs), which are suspected human carcinogens. Despite these advances, however, the role of individual human GSTs in defense against activated PAHs is poorly defined. In the present renewal application, we propose to shift emphasis from murine model to human GSTs and investigate their role in detoxification of diol epoxides. Recent studies from our laboratory and by others led us to hypothesize that the Alpha class GST isoenzymes play an important role in diol epoxide inactivation in human tissues. We propose to test this hypothesis by two different but complementary approaches. First, we will determine the kinetic constants and catalytic efficiencies for all four known Alpha class human GST isoenzymes (hGSTA1-1, hGSTA2-2, hGSTA3-3 and hGSTA4-4) toward a panel of structurally different bay- (chrysene and dibenz[a,h]anthracene) and fjord-region (benzo[c]phenanthrene and benzo[g]chrysene) type PAH diol epoxides (specific aim 1). The kinetic constants for Alpha class GSTs will be compared with those of other classes of human GSTs, including hGSTPI-1, hGSTMI-1 and hGSTTI-1, to substantiate the above hypothesis. Second, we will determine the relative contributions of human GSTs, including the Alpha class isoenzymes, for GSH conjugation of representative bay- and fjord-region diol epoxides using autopsied human liver (a major site for xenobiotic metabolism) and lung (a known target organ for PAH-induced tumorigenesis) (specific aim 2). The second objective of the present renewal application is to gain insights into the structural basis for catalytic differences in diol epoxide-GSH conjugation efficacy between human GSTs. This goal will be accomplished by two different approaches: First, we will determine the effects of mutations of key active site (H-site) residues on catalytic activity of Alpha class GSTs toward diol epoxides (specific aim 3). Second, X-ray crystallography studies are planned to elucidate the structural basis for differential substrate specificity pattern for human GSTs toward bay- and fjord-region diol epoxides (specific aim 4). In summary, the studies proposed in the present renewal application, which is a logical extension of our previous findings, will fill the gaps in our understanding of the mechanisms of diol epoxide inactivation in humans. In the long-term, this knowledge will guide us in developing strategies for prevention of PAH-induced cancers in humans.

描述（由申请人提供）：在本基金的当前资助期间，我们研究了小鼠谷胱甘肽（GSH）转移酶（GSTs）在多环芳烃（PAH）的活化代谢物（二醇环氧化物）解毒中的作用，这些多环芳烃是疑似人类致癌物。尽管有这些进展，但是，个人的GST在防御激活的多环芳烃的作用是不明确的。在目前的更新申请中，我们建议将重点从小鼠模型转移到人类GST，并研究它们在二醇环氧化物解毒中的作用。我们实验室和其他人最近的研究使我们假设α类GST同工酶在人体组织中的二醇环氧化物失活中起重要作用。我们建议通过两种不同但互补的方法来验证这一假设。首先，我们将确定所有四种已知的α类人GST同工酶（hGSTA 1 -1、hGSTA 2 -2、hGSTA 3 -3和hGSTA 4 -4）对一组结构上不同的海湾-（苯并[c]菲和苯并[g]蒽）和峡湾-区域（苯并[c]菲和苯并[g]蒽）型PAH二醇环氧化物的动力学常数和催化效率（具体目标1）。将α类GST的动力学常数与包括hGSTPI-1、hGSTMI-1和hGSTTI-1在内的其他类人GST的动力学常数进行比较，以证实上述假设。其次，我们将使用尸检的人肝脏（异生物质代谢的主要部位）和肺（PAH诱导肿瘤发生的已知靶器官）确定人GST（包括α类同工酶）对代表性海湾和峡湾地区二醇环氧化物GSH结合的相对贡献（具体目的2）。本更新申请的第二个目的是深入了解人GST之间二醇环氧化物-GSH缀合功效的催化差异的结构基础。这一目标将通过两种不同的方法来实现：首先，我们将确定关键活性位点（H-位点）残基突变对α类GST对二醇环氧化物催化活性的影响（具体目标3）。其次，计划进行X射线晶体学研究，以阐明人GST对海湾和峡湾地区二醇环氧化物的差异底物特异性模式的结构基础（具体目标4）。总之，本更新申请中提出的研究是我们先前发现的逻辑延伸，将填补我们对人类二醇环氧化物失活机制的理解空白。从长远来看，这些知识将指导我们制定预防人类PAH诱导癌症的策略。