Shining Light on the Mechanism and Regulation of Nitric Oxide Synthases

描述（由申请人提供）：一氧化氮（NO）信号失调与多种疾病有关，包括神经退行性疾病、高血压和中风、心脏病、勃起功能障碍、胃肠不适和多种癌症。NO信号在体内由一氧化氮合酶（NOS）合成开始。然而，关于NOS酶的催化和调控机制尚不清楚。NOS酶催化精氨酸氧化为NO和瓜氨酸，以氧和NADPH为辅助底物，以ng -羟精氨酸（NHA）为中间体，进行两步反应。这两个步骤都发生在NOS酶的含血红素氧化酶区域，它从含黄素还原酶区域获得电子。然而，限速电子转移掩盖了对负责精氨酸和NHA氧化的活性氧中间体的观察。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和目标2中，这些中间体将使用新的光谱技术直接观察。NOS活性受多种翻译后修饰（PTMs）的调控。阐明NOS修饰与控制NO合成之间的相互作用是一个值得研究的领域。此外，靶向ptm而非酶活性位点是治疗NOS活性失调相关疾病的正交机制。在已知的修饰中，乙酰化对内皮细胞NOS （eNOS）的抑制是最不明显的。目的3将阐明乙酰化如何与其他PTMs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。光化学诱导的NOS （iNOS）酶将被设计，其中金属标签在光激发下迅速将电子传递给血红素。这些光化学iNOS酶随后将用于“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)利用非天然底物和血红素类似物探测氧活化。非天然底物和血红素类似物将使用Aim 1和标准NOS测定中开发的技术来探索NOS酶中氧活化的结构-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。通过质谱法确定eNOS乙酰化的精确位点，进而确定乙酰化抑制eNOS的机制。乙酰化对eNOS内其他PTMs的影响也将被研究。

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.