Shining Light on the Mechanism and Regulation of Nitric Oxide Synthases

揭示一氧化氮合成酶的机制和调节

• 批准号: 8410605
• 负责人: Brian Christopher Smith
• 金额: $ 3.09万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-01 至 2012-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8410605
• 关键词: Shining; Light; Mechanism; Regulation; Nitric

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.

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，胃肠道痛和许多形式的癌症。一氧化氮合酶（NOS）的合成，没有信号传导在体内开始。然而，关于NOS酶的催化和调节机制，尚不清楚。 NOS酶将精氨酸和瓜氨酸的氧化催化为氧化剂，并在与NG-羟基氨基氨酸NHA的两步反应中使用氧和NADPH作为cosubstrate，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，该酶是从含黄素的还原酶结构域中喂养的。但是，限制电子传递掩模的速率观察活化的氧中间体负责精氨酸和NHA氧化。这些中间体的知识对于了解NOS酶的催化机制至关重要。在目标1和2中，将使用新型的光谱技术直接观察这些中间体。 NOS活性受各种翻译后修饰（PTM）的调节。阐明NOS修改以控制NO合成之间的相互作用是研究的肥沃领域。此外，靶向PTM而不是酶的活性位点是治疗与NOS活性失调相关的疾病的正交机制。在已知的修饰中，通过乙酰化对内皮NOS（ENOS）的抑制作用最少。 AIM 3将阐明乙酰化与其他PTM一起控制eNOS活动的方式。具体目的：1）直接观察到负责底物氧化的氧中间体。将设计光化学诱导的NOS（iNOS）酶，其中金属标记在光线激发后迅速向血红素传递电子。然后，这些光化学iNOS酶将用于“流动”光谱研究中，其中氧与亚铁型血红素结合，被光诱导的电子转移激活，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探测氧气激活。非天然底物和血红素类似物将使用AIM 1和标准NOS分析中开发的技术探测NOS酶中氧气激活的结构功能和热力学关系。 3）在其他翻译后修饰的背景下，确定通过乙酰化抑制eNOS的机制。 eNOS乙酰化的精确位点将通过质谱法确定，然后确定乙酰化eNOS抑制的机理。还将检查乙酰化对eNOS内其他PTM的影响。 公共卫生相关性：一氧化氮是一种与氧气相似的气体，有效的毒素，是汽车发动机和香烟烟雾产生的污染物。鉴于这种情况，人类出奇地产生一氧化氮，以在神经元，打开血管之间进行交流，并作为我们免疫反应的一部分。在此提案中，我试图了解一氧化氮是如何由一氧化氮合酶产生的，一氧化氮的产生如何在人类中控制，以及这些过程的破坏如何导致癌症，高血压和中风，胃肠道痛，心脏病，心脏病等疾病，勃起功能障碍和神经变性。