Novel GST and Detoxication of Diol Epoxides

描述（由申请人提供）：在目前的资助期内，我们研究了小鼠谷胱甘肽（GSH）转移酶（GSTs）在多环芳烃（PAHs）的活性代谢物（二醇环氧化物）解毒中的作用，多环芳烃（PAHs）被怀疑是人类致癌物。然而，尽管取得了这些进展，个体人类gst在防御活化的多环芳烃中的作用仍不明确。在目前的更新应用中，我们建议将重点从小鼠模型转移到人类模型，并研究它们在环氧二醇解毒中的作用。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环氧二醇失活中起重要作用。我们建议用两种不同但互补的方法来检验这一假设。首先，我们将确定所有四种已知的α类人类GST同工酶（hGSTA1-1、hGSTA2-2、hGSTA3-3和hGSTA4-4）对结构不同的bay- （chrysene和dibenzes [a，h]蒽）和fgulf -region （benzo[c]菲和benzo[g]chrysene）型PAH二醇环氧化合物的动力学常数和催化效率（specific aim 1）。将Alpha类gst的动力学常数与hGSTPI-1、hGSTMI-1和hgsti -1等其他类人类gst的动力学常数进行比较，以证实上述假设。其次，我们将确定人类gst的相对贡献，包括α类同工酶，用于GSH结合代表性海湾和峡湾地区的二醇环氧化物，使用尸体解剖的人类肝脏（外源代谢的主要部位）和肺（已知的靶器官，用于多环芳烃诱导的肿瘤发生）（特定目的2）。本更新申请的第二个目标是深入了解人类gst之间环氧二醇-谷胱甘肽偶联效率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对二醇环氧化物的催化活性的影响（具体目标3）。其次，计划进行x射线晶体学研究，以阐明人类gst对海湾和峡湾地区二醇环氧化物的不同底物特异性模式的结构基础（具体目标4）。综上所述，本更新申请中提出的研究是我们之前研究结果的逻辑延伸，将填补我们对人类环氧二醇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多环芳烃引起的人类癌症的策略。

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.