Mechanisms regulating oxidant release by endothelial NO synthase

项目3的长期目标是确定导致内皮一氧化氮合酶(ENOS)功能障碍的机制，并产生(除NO外)在炎症性疾病中导致氧化损伤的活性氧和氮物种(氧化剂)。我们假设eNOS有两个独特的方面对其正常功能至关重要，也使eNOS容易受到导致氧化损伤的功能障碍的影响：1)eNOS的辅因子四氢生物蝶呤(H4B)的氧化还原循环；以及2)eNOS蛋白质-蛋白质的相互作用。控制这些方面的机制尚不清楚，也是本提案的重点。我们将决定 在分子、动力学和结构水平上的机制，并最终将其转化为细胞和临床研究。目标1将测试eNOS中是否发生低效的H4B氧化还原循环，并使其易于解偶联的NO合成和氧化剂的形成。我们将：(I)量化eNOS中的H4B氧化还原循环，并开发一个动力学模型来解释氧化剂的形成，(Ii)测试eNOS突变体是否改善了H4B氧化还原循环和减少氧化剂的形成，以及(Iii)测量与增强的eNOS解偶联相关的代谢指数，以确定它们是否与人体硝化应激和心血管疾病患病率的系统性指数增加有关。目的2研究内皮型一氧化氮合酶如何通过与热休克蛋白90(HSP90)的相互作用来控制其功能。我们将：(I)验证HSP90通过影响eNOS中特定的催化步骤来改善eNOS功能的假设。(Ii)利用荧光标记、质谱学、量热法和蛋白质结晶学研究HSP90和Cav1与eNOS的结合，结合时的构象变化，以及它们对eNOS的作用机制。(Iii)设计对Cav1和HSP90具有修饰反应的eNOS定点突变体，并测试其功能；(Iv)确定当eNOS与HSP90和Akt1结合时，能够使eNOS磷酸化和激活的机制。以及(V)研究eNOS单核苷酸多态(Asp298Glu)是否与心血管风险增加相关，影响HSP90与eNOS的相互作用。总之，拟议的研究将为心血管疾病中调节eNOS功能和功能障碍的因素提供深刻的机制理解。

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.