Shining Light on the Mechanism and Regulation of Nitric Oxide Synthases

描述(申请人提供)：一氧化氮(NO)信号的失调与各种疾病有关，包括神经退行性变、高血压和中风、心脏病、勃起功能障碍、胃肠道不适和多种形式的癌症。NO信号在体内由一氧化氮合酶(NOS)合成开始。然而，关于一氧化氮合酶的催化和调节机制尚不清楚。一氧化氮合酶以氧和NADPH为共底物，以N-羟基精氨酸(NHA)为中间体，两步反应催化精氨酸氧化成NO和瓜氨酸。这两个步骤都发生在一氧化氮合酶的含血红素的氧化酶域，它从含有黄素的还原酶域获得电子。然而，限速电子转移掩盖了对精氨酸和NHA氧化负责的活性氧中间体的观察。了解这些中间产物对于理解一氧化氮合酶的催化机制至关重要。在目标1和目标2中，将使用新的光谱技术直接观察到这些中间体。一氧化氮合酶活性受多种翻译后修饰(PTM)的调节。阐明一氧化氮合酶修饰控制一氧化氮合成之间的相互作用是一个很好的研究领域。此外，靶向PTMS而不是酶活性部位是治疗与NOS活性失调相关的疾病的一种正交机制。在已知的修饰中，乙酰化对内皮型一氧化氮合酶(ENOS)的抑制作用最不典型。目标3将阐明乙酰化如何与其他PTM协同工作来控制eNOS活性。具体目标：1)直接观察负责底物氧化的氧中间体。将设计光化学诱导型一氧化氮合酶(INOS)酶，其中金属标记在光激发下将电子快速传递到血红素。这些光化学诱导型一氧化氮合酶随后将被用于流动-闪光光谱研究，其中氧与亚铁血红素结合，通过光诱导电子转移激活，并用各种光谱技术进行探测。2)使用非天然底物和血红素类似物来探测氧的活化。将使用非天然底物和血红素类似物，利用AIM 1和标准NOS分析中开发的技术，探索NOS酶中氧激活的结构-功能和热力学-动力学关系。3)在其他翻译后修饰的背景下，确定乙酰化抑制eNOS的机制。通过质谱学确定eNOS乙酰化的准确位点，进而确定乙酰化抑制eNOS的机制。此外，还将研究乙酰化对eNOS内其他PTM的影响。 与公共健康相关：一氧化氮是一种大小与氧气相似的气体，是一种强有力的毒素，也是一种由汽车发动机和香烟烟雾产生的污染物。有鉴于此，人类出人意料地产生一氧化氮，以在神经元之间进行交流，打开血管，并作为我们免疫反应的一部分。在这项提案中，我试图了解一氧化氮合酶是如何产生一氧化氮的，人类是如何控制一氧化氮的产生的，以及这些过程的中断如何导致癌症、高血压和中风、胃肠道不适、心脏病、勃起功能障碍和神经退化等疾病。

DESCRIPTION (provided by applicant): Dysregulation of nitric oxide (NO) signaling is linked to various diseases including neurodegeneration, hypertension and stroke, heart disease, erectile dysfunction, gastrointestinal distress, and many forms of cancer. NO signaling begins in vivo with its synthesis by nitric oxide synthases (NOS). However, much is unknown regarding the catalytic and regulatory mechanisms of NOS enzymes. NOS enzymes catalyze the oxidation of arginine to NO and citrulline using oxygen and NADPH as cosubstrates in a two-step reaction with NG-hydroxyarginine, NHA, as an intermediate. Both steps occur in the heme-containing oxidase domain of NOS enzymes, which is fed electrons from a flavin-containing reductase domain. However, rate limiting electron transfer masks observation of the activated oxygen intermediates responsible for arginine and NHA oxidation. Knowledge of these intermediates is crucial to understand the catalytic mechanism of NOS enzymes. In Aims 1 and 2, these intermediates will be directly observed using novel spectroscopic techniques. NOS activity is regulated by variety of post-translational modifications (PTMs). Elucidation of the interplay between NOS modifications to control NO synthesis is a fertile area for research. In addition, targeting of PTMs instead of enzyme active sites is an orthogonal mechanism to treat diseases associated with dysregulation of NOS activity. Of the known modifications, inhibition of endothelial NOS (eNOS) by acetylation is the least characterized. Aim 3 will elucidate how acetylation works in concert with other PTMs to control eNOS activity. Specific aims: 1) Directly observe oxygen intermediates responsible for substrate oxidation. Photochemical inducible NOS (iNOS) enzymes will be designed wherein metallolabels deliver electrons rapidly to the heme upon excitation with light. These photochemical iNOS enzymes will then be utilized in 'flow-flash' spectroscopic investigations, wherein oxygen is bound to a ferrous heme, activated by photoinduced electron transfer, and probed with a variety of spectroscopic techniques. 2) Use non-natural substrate and heme analogs to probe oxygen activation. Non-natural substrate and heme analogs will be used to probe the structure-function and thermodynamic-kinetic relationships of oxygen activation in NOS enzymes using the techniques developed in Aim 1 and standard NOS assays. 3) Determine the mechanism of eNOS inhibition by acetylation in the context of other post-translational modifications. The precise sites of eNOS acetylation will be determined by mass spectrometry and then the mechanism of eNOS inhibition by acetylation will be determined. The effect of acetylation on other PTMs within eNOS will also be examined. PUBLIC HEALTH RELEVANCE: Nitric oxide is a gas similar in size to oxygen, a potent toxin, and a pollutant produced by automobile engines and cigarette smoke. In light of this, humans surprisingly produce nitric oxide to communicate between neurons, to open blood vessels, and as part of our immune response. In this proposal, I seek to understand how nitric oxide is produced by nitric oxide synthases, how nitric oxide production is controlled in humans, and how disruption of these processes can lead to diseases such as cancer, hypertension and stroke, gastrointestinal distress, heart disease, erectile dysfunction, and neurodegeneration.