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

基本信息

批准号：
8128518
负责人：
Brian Christopher Smith
金额：
$ 2.21万
依托单位：
UNIVERSITY OF CALIFORNIA BERKELEY
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-08-01 至 2011-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8128518
关键词：
Acetylation Active Sites Area Arginine Automobiles Binding Biological Assay Biological Process Blood Vessels Calmodulin Chemicals Chemistry Citrulline Cutaneous Cytochrome P450 Disease Distress Drug Delivery Systems Electron Transport Electrons Enzymes Erectile dysfunction Flavins Gases Goals Heart Diseases Heme Human Hydroxylation Hypertension Immune response Investigation Kinetics Knowledge Lead Light Link Malignant Neoplasms Masks Mass Spectrum Analysis Modification NADP Nature Nerve Degeneration Neurons Nitric Oxide Nitric Oxide Synthase Nitrosation Oxidases Oxidoreductase Oxygen Phosphorylation Post-Translational Protein Processing Process Production Protein Acetylation Reaction Regulation Research Signal Transduction Signaling Molecule Site Stroke Structure Sulfhydryl Compounds T-Cell Lymphoma Techniques Thermodynamics Toxin Vorinostat Work analog cigarette smoking design feeding gastrointestinal human NOS2A protein human NOS3 protein human disease hypertensive heart disease in vivo myristoylation novel oxidation palmitoylation pollutant public health relevance

项目摘要

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、胃肠道不适和多种癌症。 NO 信号传导在体内始于一氧化氮合酶 (NOS) 的合成。然而，对于 NOS 酶的催化和调节机制尚不清楚。 NOS 酶使用氧和 NADPH 作为共底物，以 NG-羟基精氨酸 (NHA) 作为中间体，在两步反应中催化精氨酸氧化为 NO 和瓜氨酸。这两个步骤都发生在 NOS 酶的含血红素氧化酶结构域中，该结构域由含黄素的还原酶结构域提供电子。然而，电子转移速率限制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 2 中，将使用新型光谱技术直接观察这些中间体。 NOS 活性受多种翻译后修饰 (PTM) 调节。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 而不是酶活性位点是治疗与 NOS 活性失调相关疾病的正交机制。在已知的修饰中，通过乙酰化抑制内皮型一氧化氮合酶（eNOS）的特征最少。目标 3 将阐明乙酰化如何与其他 PTM 协同作用来控制 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。然后，这些光化学 iNOS 酶将用于“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) 使用非天然底物和血红素类似物来探测氧的活化。使用目标 1 和标准 NOS 测定中开发的技术，将使用非天然底物和血红素类似物来探测 NOS 酶中氧活化的结构-功能和热力学-动力学关系。 3) 确定在其他翻译后修饰背景下通过乙酰化抑制 eNOS 的机制。通过质谱法确定eNOS乙酰化的精确位点，然后确定乙酰化抑制eNOS的机制。还将检查乙酰化对 eNOS 内其他 PTM 的影响。公共健康相关性：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，令人惊讶的是，人类会产生一氧化氮来在神经元之间进行通讯，打开血管，并作为我们免疫反应的一部分。在这个提案中，我试图了解一氧化氮合酶是如何产生一氧化氮的，人类如何控制一氧化氮的产生，以及这些过程的破坏如何导致癌症、高血压和中风、胃肠道不适、心脏病等疾病、勃起功能障碍和神经退行性疾病。