eNOS-actin Interaction and Oxygen in Lung Endothelium

肺内皮细胞中的 eNOS-肌动蛋白相互作用和氧

• 批准号: 7842045
• 负责人: YUNCHAO SU
• 金额: $ 36.75万
• 依托单位: AUGUSTA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-01 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7842045
• 关键词: Actins; Adenoviruses; Arginine; Binding; Binding Sites; Biology; Blood Vessels; Cell Proliferation; Chronic Obstructive Airway Disease; Cytochrome c Reductase; Cytoskeleton; Data; Electron Transport; Endothelial Cells; Endothelium; Enzyme Kinetics; Gene Transfer; Hyperoxia; Hypoxia; Lead; Lung; Lung diseases; Mediating; Microinjections; Nitric Oxide; Oxidoreductase; Oxygen; Oxygen Therapy Care; Oxygen measurement, partial pressure, arterial; Patients; Peptides; Platelet aggregation; Play; Production; Proteins; Pulmonary Heart Disease; Pulmonary Hypertension; Reaction; Regulation; Reporting; Research; Role; Small Interfering RNA; Smooth Muscle Myocytes; Techniques; Technology; Testing; Vasodilation; angiogenesis; cytochrome c; ferricyanide reductase; human NOS3 protein; interdisciplinary approach; novel; protein aminoacid sequence; protein protein interaction; public health relevance; pulmonary artery endothelial cell; research study; yeast two hybrid system

DESCRIPTION (provided by applicant): Endothelial nitric oxide synthase (eNOS) is tightly regulated by a variety of transcriptional, posttranscriptional, and posttranslational mechanisms. We have found that eNOS is associated with the actin protein and that this association increases eNOS activity. We propose a novel hypothesis that eNOS-actin association regulates electron transfer of eNOS resulting in increased catalytic activity and that alterations in eNOS-actin interaction mediate the regulation of eNOS in lung endothelium exposed to different oxygen tensions. Aim #1: Determine whether actin interacts with the putative actin binding sequences (ABSs) in the eNOS protein resulting in increased electron transfer and catalytic activity of eNOS. We will use a yeast two-hybrid system and the peptides with sequences of the three putative ABSs of eNOS to pinpoint the actin-binding site in the eNOS protein. We will evaluate the effects of ABS peptides of eNOS on eNOS activity, eNOS-actin interaction, the enzyme kinetics, and on cytochrome c and ferricyanide reductase activities in the mixture of purified eNOS and actin to clarify the mechanism for increased eNOS activity by actin association. Aim #2: Determine whether alterations of eNOS activity in lung endothelium exposed to different oxygen tensions are due to changes in eNOS-actin association. We will manipulate the availability of actin in endothelium using siRNA and adenovirus gene transfer technology and introduce the ABS peptides of eNOS into endothelial cells to block eNOS-actin interaction using microinjection technique. Then we will observe hypoxia/hyperoxia-induced alterations in eNOS activity, the actin cytoskeleton, eNOS-actin association, NO release, and endothelium-dependent vasorelaxation. The successful completion of these aims will advance our understanding of the biology of NO, and in turn, have major implications for pulmonary diseases characterized by impaired eNOS activity and vascular NO production, such as primary and secondary pulmonary hypertension, COPD, and cor pulmonale. Determination of the effect of short-term hyperoxia will help to advance our understanding of the mechanism of oxygen therapy and will lead to better management strategy for patients receiving supplemental oxygen therapy. PUBLIC HEALTH RELEVANCE: This proposal is to study a novel hypothesis that eNOS-actin association regulates electron transfer of eNOS resulting in increased catalytic activity and that alterations in eNOS-actin interaction mediate the regulation of eNOS in lung endothelium exposed to different oxygen tensions. Proof of the role of eNOS-actin interaction in hypoxia/hyperoxia-induced alteration of eNOS function would advance our understanding of the biology of NO, and in turn, have major implications for pulmonary diseases characterized by impaired eNOS activity and vascular NO production, such as primary and secondary pulmonary hypertension, COPD, and cor pulmonale. Determination of the effect of short-term hyperoxia will help to advance our understanding of the mechanism of oxygen therapy and will lead to better management strategy for patients receiving supplemental oxygen therapy.

描述（由申请人提供）：内皮一氧化氮合酶（eNOS）受到多种转录、转录后和翻译后机制的严格调控。我们发现 eNOS 与肌动蛋白相关，并且这种关联增加了 eNOS 活性。我们提出了一个新的假设，即 eNOS-肌动蛋白关联调节 eNOS 的电子转移，从而导致催化活性增加，并且 eNOS-肌动蛋白相互作用的改变介导暴露于不同氧张力的肺内皮中 eNOS 的调节。目标#1：确定肌动蛋白是否与 eNOS 蛋白中推定的肌动蛋白结合序列 (ABS) 相互作用，从而导致 eNOS 的电子转移和催化活性增加。我们将使用酵母双杂交系统和具有 eNOS 三个假定 ABS 序列的肽来精确定位 eNOS 蛋白中的肌动蛋白结合位点。我们将评估纯化的 eNOS 和肌动蛋白混合物中 eNOS 的 ABS 肽对 eNOS 活性、eNOS-肌动蛋白相互作用、酶动力学以及对细胞色素 c 和铁氰化物还原酶活性的影响，以阐明通过肌动蛋白结合增加 eNOS 活性的机制。目标#2：确定暴露于不同氧张力的肺内皮细胞中 eNOS 活性的变化是否是由于 eNOS-肌动蛋白关联的变化所致。我们将利用siRNA和腺病毒基因转移技术操纵内皮细胞中肌动蛋白的可用性，并利用显微注射技术将eNOS的ABS肽引入内皮细胞以阻断eNOS与肌动蛋白的相互作用。然后我们将观察缺氧/高氧诱导的 eNOS 活性、肌动蛋白细胞骨架、eNOS-肌动蛋白关联、NO 释放和内皮依赖性血管舒张的变化。这些目标的成功完成将增进我们对 NO 生物学的理解，进而对以 eNOS 活性和血管 NO 产生受损为特征的肺部疾病（如原发性和继发性肺动脉高压、慢性阻塞性肺病和肺心病）产生重大影响。确定短期高氧的影响将有助于加深我们对氧疗机制的理解，并将为接受补充氧疗的患者提供更好的管理策略。 公共健康相关性：本提案旨在研究一种新的假设，即 eNOS-肌动蛋白关联调节 eNOS 的电子转移，从而导致催化活性增加，并且 eNOS-肌动蛋白相互作用的改变介导暴露于不同氧张力的肺内皮中 eNOS 的调节。证明 eNOS-肌动蛋白相互作用在缺氧/高氧诱导的 eNOS 功能改变中的作用将促进我们对 NO 生物学的理解，反过来，对以 eNOS 活性和血管 NO 产生受损为特征的肺部疾病（如原发性和继发性肺动脉高压、慢性阻塞性肺病和肺心病）具有重大意义。确定短期高氧的影响将有助于加深我们对氧疗机制的理解，并将为接受补充氧疗的患者提供更好的管理策略。