Glutathione S-Transferases: Novel Regulators of Cellular Stress and Apoptosis

描述(申请人提供)：谷胱甘肽S转移酶(GSTs)是一个在外源生物解毒过程中起重要作用的酶家族。由于它们催化已知致癌物的失活，它们提供了一种对抗癌变的防御；然而，它们对癌症化疗的耐药性的形成有很大贡献，因为肿瘤中GST水平增加，这种酶代谢关键的抗癌药物。与其他蛋白质的相互作用是GSTs的一个新角色，它产生了对多种细胞功能的调节。因此，GSTs促进细胞对氧化应激的反应，因为GSTpi激活抗氧化酶1-半胱氨酸过氧化还蛋白(1-Cys Prx)；GSTs通过与Jun N末端激酶(JNK)和凋亡信号调节蛋白1(ASK1)相互作用影响细胞信号转导。我们将与最丰富的哺乳动物GSTs中的pi、Mu和Alpha类GSTs的代表一起解决以下问题：1)GSTpi激活1-Cys Prx的结构和化学基础是什么？将分离GST与1-Cys Prx之间的异二聚体络合物，测定其谷胱甘肽S转移酶和过氧化还蛋白活性的动力学性质，并测定其物理化学和底物结合特性。2)GSTpi是如何抑制应激激活的蛋白激酶JNK的，该复合体的谷胱甘肽S转移酶的活性是什么？这种效应是pi类GST所特有的，还是抑制JNK是GST的一种普遍特性？GST-JNK络合物将被分离出来，以严格表征其催化和生物物理性质。3)MU类GST抑制信号转导蛋白凋亡信号调节激酶1的分子基础是什么？对于ASK1和GST(野生型和突变型)形成的ASK1-GST复合体，我们将测量其分子量、构象和氧化还原状态，以阐明ASK1介导的细胞凋亡受到抑制的原因以及复合体形成对GST活性的影响。确定谷胱甘肽和/或外源底物是否参与GST的蛋白质-蛋白质功能是很重要的。这些研究可能导致合理的药物设计，通过抑制特定的GST位点来延长抗癌药物的寿命，而不会对其其他功能位点产生不利影响。

DESCRIPTION (provided by applicant): Glutathione S-transferases (GSTs) constitute a family of enzymes important in the detoxification of xenobiotics. They provide a defense against carcinogenesis, since they catalyze inactivation of known carcinogens; yet they contribute significantly to the development of resistance to cancer chemotherapy, since GST levels increase in tumors and the enzyme metabolizes key anticancer drugs. Interaction with other proteins is a new role for GSTs which yields regulation of diverse cellular functions. Thus, GSTs promote the cellular response to oxidative stress, since GSTpi activates the anti-oxidant enzyme 1-Cys peroxiredoxin (1-Cys Prx); and GSTs influence cell signaling through interactions with Jun N-terminal Kinase (JNK) and Apoptosis Signal-Regulating Kinase 1 (ASK1). With representatives of the pi, mu and alpha class GSTs, the most abundant mammalian GSTs, we will address the following questions: 1) What is the structural and chemical basis of activation by GSTpi of 1-Cys Prx? The heterodimeric complex between GST and 1-Cys Prx will be isolated, its kinetic properties for glutathione S-transferase and peroxiredoxin activities will be determined, and its physicochemical, as well as its substrate binding characteristics will be measured. 2) How does GSTpi inhibit the stress-activated kinase JNK, and what is the glutathione S-transferase activity of the complex? Is this effect specific for pi class GST or is inhibition of JNK a general property of GSTs? GST-JNK complexes will be isolated for rigorous characterization of its catalytic and biophysical properties. 3) What is the molecular basis of inhibition by mu class GST of the signal transduction protein Apoptosis Signal-Regulating Kinase 1? For isolated ASK1-GST complexes formed from ASK1 and GST (both wild-type and mutant), the molecular weight, conformation and re-dox state will be measured to elucidate the cause of inhibition of ASK1-mediated apoptosis and the effect of complex formation on GST activity. It is important to ascertain whether the glutathione and/or xenobiotic substrate sites are involved in the protein-protein functions of GST. These studies may lead to the rational design of pharmaceutical agents to prolong the lifetime of anticancer drugs by inhibiting particular GST sites without adversely affecting its other functional sites.