Glutathione S-Transferases and Oxidative Stress

谷胱甘肽 S-转移酶和氧化应激

• 批准号: 7556363
• 负责人: WILLIAM M ATKINS
• 金额: $ 27.71万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-01-10 至 2011-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7556363
• 关键词: 4 hydroxynonenal; ABCC1 gene; Active Sites; Affect; Alzheimer' s Disease; Atherosclerosis; Behavior; Binding; Biological; COSY; Carbon; Catalysis; Cataract; Cells; Complex; Computer Simulation; Crystallography; Devices; Disease; Disease Progression; Drug Metabolic Detoxication; Engineering; Enzymatic Biochemistry; Enzymes; Equilibrium; Evolution; Exhibits; Face; Fluorescence; Genetic Transcription; Glutathione; Glutathione S-Transferase; Goals; Grant; Hepatic; Homeostasis; Human; Individual; Isomerism; Knowledge; Libraries; Ligands; Lipids; Malignant Neoplasms; Mass Spectrum Analysis; Measures; Mediating; Metabolism; Modeling; Molecular; Monitor; Motion; Multi-Drug Resistance; Mutagenesis; NOESY; Oxidative Stress; Parkinson Disease; Pathway interactions; Pharmaceutical Preparations; Prostaglandins; Protein Dynamics; Protein Isoforms; Proteins; Pump; ROESY; Recruitment Activity; Relative (related person); Reporting; Response Elements; Role; Route; Series; Specificity; Stress; Structure; Substrate Specificity; Time; Toxic effect; analog; base; biological adaptation to stress; enantiomer; mutant; peroxidation; pi bond; symporter

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.

描述（由申请人提供）：典型的胞质谷胱甘肽 S-转移酶 (GST) 具有高度特异性、异构体依赖性、抗氧化应激功能，可调节疾病的进展，包括动脉粥样硬化、帕金森病、阿尔茨海默病、白内障形成、药物引起的肝毒性，或癌症。该提案旨在填补我们在分子水平上对 GST 依赖性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 为此提供了一个极好的模型。 GSTA4-4 催化谷胱甘肽 (GSH) 与氧化应激的几种脂质或前列腺素过氧化产物结合，例如 4-羟基壬烯醛 (HNE)、13-Oxo 或异前列腺素 (Isops)。尽管 HNE 和 Isops 由于其亲电子反应性而在高浓度下可能有毒，但它们对于调节多种应激反应元件转录的稳态机制也至关重要。 HNE 和异前列腺素都包含手性中心和前手性中心，导致 GSH 缀合产生多种可能的非对映体产物。然而，人 GSTA4-4 对于底物和产物的立体化学选择性尚未确定。此外，初步建模表明，GSTA4-4 已经招募了典型 A 类 GST 的催化 Tyr-9 和 Arg-15 来实现新的目的； GSTA4-4 中的这些残基可以为两种 HNE 对映体提供识别，以允许底物立体混杂。因此，具体目标 1 和 2 包括完全确定 GSTA4-4 催化 GSH 与 HNE 缀合的立体化学过程，以及与 GSTA4-4 复合的各个 HNE 对映体以及与非反应性 GSH 的三元复合物的晶体学分析模拟。结合这些目标，将确定谷胱甘肽缀合物 (GS-X) 转运蛋白 MRP2 和 RLIP76 的立体选择性，并与 GSTA4-4 进行比较。这样的比较探讨了 GSTA4-4 和转运蛋白以匹配的立体选择性共同进化的可能性。具体目标 3 探讨了混杂的原型解毒酶 GSTA1-1 与底物特异性 GSTA4-4 的差异动力学，两者具有几乎相同的晶体结构。该目标包括在存在和不存在几种结构不相关的底物的情况下比较 GSTA1-1 和 A4-4 的动力学。在Specific Aim 3中，GSTA1-1和A4-4的突变体将逐渐反转其相对特异性和混杂性，对于这一系列突变体，将通过H/D交换质谱和荧光寿命分布分析来测量局部蛋白质动态。这些研究探讨了配体诱导的构象变化或“诱导配合”可用于实现底物混杂以及底物选择性的可能性。总之，这些研究增加了我们对 GST 依赖性氧化应激反应的了解，以及我们对有助于特异性与混杂性的蛋白质结构/功能/动力学的理解。