Glutathione S-Transferases and Oxidative Stress

描述(申请人提供)：典型的胞浆谷胱甘肽S转移酶(GSTs)具有高度特异性、同工型依赖的抗氧化应激功能，可调节动脉粥样硬化、帕金森病、阿尔茨海默病、白内障形成、药物诱导的肝毒性或癌症等疾病的进展。这一建议旨在填补我们在分子水平上对GST依赖的氧化应激反应的理解空白，并了解蛋白质动力学在底物混杂解毒酶中的作用，GST为此提供了一个很好的模型。GSTA4-4催化谷胱甘肽(GSH)结合成几种脂质或前列腺素氧化应激的过氧化产物，如4-羟基壬烯醛(HNE)、13-氧代(13-oxo)或异前列腺素(Isops)。尽管HNE和Isop在高浓度时可能因其亲电反应而有毒，但它们也是调节几个应激反应元件转录的动态平衡机制的关键。HNE和异前列腺素都包括手性中心和前手性中心，导致多种可能的GSH结合的非对映异构体产物。然而，人GSTA4-4对底物和产物的立体化学选择性尚未确定。此外，初步的模拟表明，GSTA4-4已经招募了典型的A类GSTs的催化Tyr-9和Arg-15用于新的目的；GSTA4-4中的这些残基可能对这两个HNE对映体提供识别，从而允许底物立体混杂。因此，特定的目标1和2包括完全确定GSTA4-4催化的GSH与HNE偶联的立体化学过程，以及对与GSTA4-4络合的单个HNE对映体以及与非反应性GSH类似物的三元络合物的结晶学分析。结合这些目标，将测定谷胱甘肽结合物(GS-X)转运体MRP2和RLIP76的立体选择性，并与GSTA4-4进行比较。这种比较探索了GSTA4-4和转运蛋白以匹配的立体选择性共同进化的可能性。特定目标3探索了混杂原型解毒酶GSTA1-1和底物特异性GSTA4-4的差异动力学，它与GSTA4-4具有几乎相同的晶体结构。这一目标包括比较GSTA1-1和A4-4在存在和不存在几种结构无关底物的情况下的动力学。针对GSTA1-1和A4-4的3个突变体，逐步逆转其相对专一性和杂交性，并对这一系列突变体进行H/D交换质谱分析和荧光寿命分布分析。这些研究探索了配体诱导的构象变化或“诱导配对”可用于实现底物混杂和底物选择性的可能性。总之，这些研究增加了我们对依赖GST的氧化应激反应的了解，以及我们对导致特异性与混杂的蛋白质的结构/功能/动力学的理解。

DESCRIPTION (provided by applicant): The canonical cytosolic glutathione S-transferases (GSTs) contribute highly specific, isoform-dependent, antioxidative stress functions that modulate progression of diseases including atherosclerosis, Parkinson's disease, Alzheimer's disease, cataract formation, drug-induced hepatic toxicity, or cancer. This proposal aims to fill gaps in our understanding at the molecular level of GST-dependent oxidative stress responses and to understand the role of protein dynamics in substrate promiscuous detoxification enzymes, for which the GSTs provide an excellent model. GSTA4-4 catalyzes the conjugation of glutathione (GSH) to several lipid or prostaglandin peroxidation products of oxidative stress, such as 4-hydroxynonenal (HNE), 13-Oxo, or isoprostanoids (Isops). Although HNE and Isops may be toxic at high concentrations due to their electrophilic reactivity, they also are critical to homeostatic mechanisms wherein they regulate transcription of several stress response elements. Both HNE and isoprostanoids include chiral centers and prochiral centers leading to multiple possible diasatereomeric products of GSH conjugation. However, the stereochemical selectivity of human GSTA4-4 with respect to substrate and product has not been determined. Moreover, preliminary modeling suggests that GSTA4-4 has recruited the catalytic Tyr-9 and Arg-15 of the canonical A-class GSTs for a new purpose; these residues in GSTA4-4 may provide recognition for both HNE enantiomers, to allow substrate stereopromiscuity. Therefore, Specific Aims 1 and 2 encompass complete determination of the stereochemical course of GSTA4-4 catalyzed conjugation of GSH to HNE, as well as crystallographic analysis of the individual HNE enantiomers complexed with GSTA4-4, and in the ternary complexes with a nonreactive GSH analog. In conjunction with these aims, the stereoselectivity of the glutathione conjugate (GS-X) transporters MRP2 and RLIP76 will be determined and compared to GSTA4-4. Such a comparison explores the possibility that GSTA4-4 and transporters co-evolved with matching stereoselectivity. Specific Aim 3 explores the differential dynamics of the promiscuous archetypal detoxification enzyme GSTA1-1 with the substrate specific GSTA4-4, to which it shares a nearly identical crystal structure. This aim includes a comparison of the dynamics of GSTA1-1 and A4-4 in the presence and absence of several structurally unrelated substrates. In Specific Aim 3 mutants of GSTA1-1 and A4-4 will be made to incrementally invert their relative specificity and promiscuity, and for this series of mutants, local protein dynamics will be measured by H/D exchange mass spectrometry and fluorescence lifetime distribution analysis. These studies explore the possibility that ligand-induced conformational change, or "induced fit" can be used to achieve substrate promiscuity as well as substrate selectivity. Together, these studies add to our knowledge of GST-dependent oxidative stress responses as well as our understanding of the structure/function/dynamics of proteins that contribute to specificity vs. promiscuity.