Shining Light on the Mechanism and Regulation of Nitric Oxide Synthases

描述（由申请人提供）：一氧化氮（NO）信号传导的失调与各种疾病有关，包括神经变性、高血压和中风、心脏病、勃起功能障碍、胃肠道不适和许多形式的癌症。NO信号在体内开始于其通过一氧化氮合酶（NOS）的合成。然而，关于NOS酶的催化和调节机制还有很多未知之处。NOS酶催化精氨酸氧化为NO和瓜氨酸，使用氧气和NADPH作为共底物，在两步反应中使用NG-羟基精氨酸（NHA）作为中间体。这两个步骤都发生在NOS酶的含血红素的氧化酶结构域中，该结构域由含黄素的还原酶结构域提供电子。然而，限速电子转移掩模观察负责精氨酸和NHA氧化的活性氧中间体。了解这些中间体对于理解NOS酶的催化机制至关重要。在目标1和2中，将使用新的光谱技术直接观察这些中间体。NOS活性受多种翻译后修饰（PTMs）的调节。阐明NOS修饰之间的相互作用以控制NO合成是一个丰富的研究领域。此外，靶向PTM而不是酶活性位点是治疗与NOS活性失调相关的疾病的正交机制。在已知的修饰中，乙酰化对内皮NOS（eNOS）的抑制是最少表征的。目的3将阐明乙酰化如何与其他PTM协同作用以控制eNOS活性。具体目的：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NOS（iNOS）酶，其中金属标记物在用光激发时将电子快速递送至血红素。然后，这些光化学iNOS酶将被用于“流动闪光”光谱调查，其中氧被绑定到亚铁血红素，通过光诱导电子转移激活，并探测与各种光谱技术。2)使用非天然底物和血红素类似物来探测氧活化。非天然底物和血红素类似物将用于探测NOS酶中氧活化的结构-功能和代谢动力学关系，使用Aim 1和标准NOS测定中开发的技术。3)确定在其他翻译后修饰的背景下通过乙酰化抑制eNOS的机制。将通过质谱法确定eNOS乙酰化的精确位点，然后确定乙酰化抑制eNOS的机制。还将检查乙酰化对eNOS内的其他PTM的影响。 公共卫生相关性：一氧化氮是一种大小与氧气相似的气体，是一种强效毒素，也是汽车发动机和香烟烟雾产生的污染物。有鉴于此，人类令人惊讶地产生一氧化氮来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。在这个提议中，我试图了解一氧化氮是如何由一氧化氮合酶产生的，一氧化氮的产生是如何在人类中控制的，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行性疾病等疾病。

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.