Mechanisms regulating oxidant release by endothelial NO synthase

项目3的长期目标是确定导致内皮NO合酶（eNOS）功能障碍并产生（除NO外）活性氧和氮物质（氧化剂）的机制，这些活性氧和氮物质在炎症性疾病中引起氧化损伤。我们假设eNOS的两个独特方面对其正常功能至关重要，并且也使eNOS易于发生导致氧化损伤的功能障碍：1）其辅因子四氢生物蝶呤（H4 B）的eNOS氧化还原循环;和2）eNOS蛋白-蛋白相互作用。控制这些方面的机制尚不清楚，这是本提案的重点。我们将确定 在分子、动力学和结构水平上研究其机制，并最终将其转化为细胞和临床研究。AIM 1将测试eNOS中是否发生低效的H4 B氧化还原循环，并使其倾向于解偶联NO合成和氧化剂形成。我们将：（i）量化eNOS中的H4 B氧化还原循环并开发动力学模型以解释氧化剂形成，（ii）测试eNOS突变体的改善的H4 B氧化还原循环和较少的氧化剂形成，以及（iii）测量与增强的eNOS解偶联相关的代谢指数以确定它们是否与硝化应激的增加的系统指数和人类心血管疾病的患病率相关。目的2研究eNOS与热休克蛋白90（HSP 90）的相互作用对eNOS功能的影响。我们将：（i）测试HSP 90通过影响eNOS中的特定催化步骤来改善eNOS功能的假设。(ii)利用荧光标记，质谱，量热法和蛋白质晶体学研究HSP 90和Cav 1与eNOS的结合，伴随结合的构象变化，以及它们对eNOS的作用机制。(iii)设计eNOS的位点特异性突变体，其具有对Cavl和HSP 90的修饰的响应，并测试它们的功能。（iv）确定当与HSP 90和激酶Aktl结合在三聚体复合物中时能够磷酸化和激活eNOS的机制。以及（v）研究与心血管风险增加相关的eNOS（Asp 298 Glu）中的单核苷酸多态性是否影响HSP 90与eNOS的相互作用。总的来说，拟议的研究将提供一个深刻的机制的理解，在心血管疾病中的eNOS功能和功能障碍的调节因素。

The long-term goal of Project 3 is to define mechanisms that cause endothelial NO synthase (eNOS) to dysfunction and generate (in addition to NO) reactive oxygen and nitrogen species (oxidants) that cause oxidative damage in inflammatory diseases. We hypothesize that two unique aspects of eNOS are critical for its proper function and also predispose eNOS to be susceptible to dysfunction leading to oxidative damage: 1) eNOS redox cycling of its cofactor tetrahydrobiopterin (H4B); and 2) eNOS protein-protein interactions. Mechanisms that control these aspects are unclear and are the focus of this proposal. We will determine mechanisms at a molecular, kinetic, and structural level and ultimately translate them to cellular and clinical studies. AIM 1 will test if inefficient H4B redox cycling occurs in eNOS and predisposes it to uncoupled NO synthesis and oxidant formation. We will: (i) Quantify H4B redox cycling in eNOS and develop a kinetic model to explain oxidant formation, (ii) Test eNOS mutants for improved H4B redox cycling and less oxidant formation, and (iii) Measure metabolic indices associated with enhanced eNOS uncoupling to determine if they are linked with increased systemic indices of nitrative stress and prevalence for cardiovascular disease in humans. AIM 2 will investigate how eNOS function is controlled by interactions with heat shock protein 90 (HSP90). We will: (i) Test the hypothesis that HSP90 improves eNOS function by affecting specific catalytic steps in eNOS. (ii) Utilize fluorescence labeling, mass spectrometry, calorimetry, and protein crystallography to investigate HSP90 and Cav1 binding to eNOS, conformational changes that accompany binding, and mechanism for their effects on eNOS. (iii) Design site-specific mutants of eNOS with modified responses to Cavl and HSP90 and test their function, (iv) Determine mechanisms that enable phosphorylation and activation of eNOS when bound in a trimeric complex with HSP90 and the kinase Akt1. and (v) Investigate if a single nucleotide polymorphism in eNOS (Asp298Glu) associated with increased cardiovascular risk impacts HSP90 interactions with eNOS. Collectively, the proposed studies will provide a deep mechanistic understanding of factors regulating eNOS fucntion and dysfunction in cardiovascular disease.