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

描述（由申请人提供）：谷胱甘肽S-转移酶（GST）是一个在异生物质解毒中起重要作用的酶家族。它们提供了对致癌作用的防御，因为它们催化已知致癌物的失活;然而，它们显著促进了对癌症化疗的耐药性的发展，因为GST水平在肿瘤中增加，并且该酶代谢关键的抗癌药物。与其他蛋白质的相互作用是GST的一个新的角色，它产生了不同的细胞功能的调节。因此，GST促进细胞对氧化应激的反应，因为GST β 1激活抗氧化酶1-Cys过氧化物氧还蛋白（1-Cys Prx）; GST通过与Jun N-末端激酶（JNK）和凋亡信号调节激酶1（ASK 1）相互作用影响细胞信号传导。与代表的pi，μ和α类GST，最丰富的哺乳动物GST，我们将解决以下问题：1）是什么结构和化学基础的激活1-半胱氨酸蛋白酶的GST π？将分离GST和1-Cys Prx之间的异二聚体复合物，确定其谷胱甘肽S-转移酶和过氧化物氧还蛋白活性的动力学特性，并测量其物理化学及其底物结合特征。2)GSTpi如何抑制应激激活激酶JNK，复合物的谷胱甘肽S-转移酶活性是什么？这种效应是否对pi类GST具有特异性，或者抑制JNK是GST的一般特性？GST-JNK复合物将被分离，以严格表征其催化和生物物理特性。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？对于由ASK 1和GST（野生型和突变型）形成的分离的ASK 1-GST复合物，将测量分子量、构象和氧化还原状态，以阐明抑制ASK 1介导的细胞凋亡的原因以及复合物形成对GST活性的影响。重要的是要确定是否谷胱甘肽和/或外源性底物位点参与GST的蛋白质-蛋白质功能。这些研究可能会导致合理设计的药剂，以延长抗癌药物的寿命，抑制特定的GST网站，而不会产生不利影响的其他功能网站。

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.