Regulation of Endothelial Nitric Oxide Synthase mRNA stability

内皮一氧化氮合酶 mRNA 稳定性的调节

• 批准号: 8389616
• 负责人: Jianxin Sun
• 金额: $ 36.89万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 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 2015-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8389616
• 关键词: 3' Untranslated Regions; Adenoviruses; Adhesions; Affect; Affinity; Affinity Chromatography; Amino Acid Sequence; Atherosclerosis; Binding; Binding Proteins; Biological Assay; Blood Vessels; Cardiovascular Diseases; Cell Culture Techniques; Chronic; Chronic Kidney Failure; Coenzyme A; Complex; Coronary Arteriosclerosis; Data; Development; Diabetes Mellitus; Dominant-Negative Mutation; Down-Regulation; Endothelial Cells; Enzymes; Estrogens; Exercise; Functional disorder; Growth; Heart failure; Homeostasis; Human; Hypertension; Hypoxia; Immunoprecipitation; Lead; Leukocytes; Lipopolysaccharides; Maintenance; Mass Spectrum Analysis; Mediating; Messenger RNA; Modeling; Molecular; Nitric Oxide; Oryctolagus cuniculus; Oxidoreductase; Pathway interactions; Peptide Sequence Determination; Peripheral arterial disease; Phosphorylation; Physiological; Platelet aggregation; Play; Polypyrimidine Tract-Binding Protein; Proteins; RNA; Regulation; Rho-associated kinase; Ribonucleoproteins; Role; Small Interfering RNA; Stimulus; TNF gene; Testing; Therapeutic Agents; Translations; Tumor Necrosis Factor-alpha; Vascular Diseases; Vasodilation; base; cell growth; crosslink; cytokine; gel mobility shift assay; human EEF1A1 protein; human NOS3 protein; human disease; hypercholesterolemia; in vivo; inhibitor/antagonist; insight; mRNA Decay; mRNA Stability; new therapeutic target; novel therapeutics; overexpression; oxidized low density lipoprotein; prevent; protein complex; protein expression; protein protein interaction; public health relevance; rho; shear stress; vascular smooth muscle cell proliferation

DESCRIPTION (provided by applicant): Nitric oxide (NO), produced by endothelial nitric oxide synthase (eNOS), is critical to the maintenance of vascular homeostasis through promoting vasodilation and inhibiting vascular smooth muscle cells proliferation, platelets aggregation, and leukocyte adhesion. Although eNOS was initially considered to be constitutive, it was later demonstrated that several pathophysiological stimuli, such as shear stress, chronic exercise, and the subconfluent growth state, upregulate eNOS expression. In contrast, cytokines including tumor necrosis factor-alpha, hypoxia, and high concentrations of oxidized LDL decrease eNOS levels, which may contribute to the endothelial dysfunction associated with many cardiovascular diseases such as atherosclerosis and heart failure. For many of these stimuli, modulation of eNOS mRNA stability plays an essential role in controlling eNOS expression. Accumulating evidence indicates that the binding of cytosolic protein(s) to eNOS 3' untranslated region (3'-UTR) plays a critical role in the regulation of eNOS mRNA stability. However, the identity of these proteins remains to be identified. By using RNA affinity purification and protein sequencing by mass spectrometry, we have revealed that translation elongation factor-1 alpha (eEF1A) and polypyrimidine tract-binding protein (PTB) specifically bind to eNOS 3'-UTR, potentially regulating eNOS mRNA stability in human endothelial cells. This proposal is to continue to explore, in depth, the mechanisms by which PTB and eEF1A1 regulate eNOS mRNA stability in cell culture and in vivo. Aim 1 will characterize the physical and functional interactions of eNOS mRNA 3'-UTR with PTB and identify their interacting domains by RNA gel mobility shift assays and UV-cross linking assays. Aim 2 will investigate the molecular mechanisms regulating the eNOS mRNA 3'-UTR ribonucleoprotein complex formation. Emphasis will placed on the roles of the Rho/ROCK pathway and protein-protein interactions in the regulation of eNOS 3'-UTR ribonucleoprotein complex formation and eNOS mRNA stability. Furthermore, aim 3 will investigate whether disruption of the eNOS 3'-UTR/eEF1A1/PTB complex will prevent eNOS mRNA destabilization induced by TNF-1 in endothelial cells and whether eEF1A1 and PTB participate in the development of endothelial dysfunction in a rabbit model of hypercholesterolemia. These proposed studies will provide greater insights into the mechanisms of how eNOS expression is regulated under physiological and pathological conditions. It's hoped that the results obtained from the proposed studies will lead to novel therapeutic targets for treatment of cardiovascular diseases.

描述（申请人提供）：一氧化氮（NO）由内皮一氧化氮合酶（eNOS）产生，通过促进血管舒张和抑制血管平滑肌细胞增殖、血小板聚集和白细胞粘附，对维持血管稳态至关重要。虽然 eNOS 最初被认为是组成型的，但后来证明，一些病理生理刺激，如剪切应力、慢性运动和亚汇合生长状态，会上调 eNOS 表达。相反，包括肿瘤坏死因子-α、缺氧和高浓度氧化 LDL 在内的细胞因子会降低 eNOS 水平，这可能导致与许多心血管疾病（如动脉粥样硬化和心力衰竭）相关的内皮功能障碍。对于许多这些刺激，eNOS mRNA 稳定性的调节在控制 eNOS 表达中起着至关重要的作用。越来越多的证据表明，胞质蛋白与 eNOS 3' 非翻译区 (3'-UTR) 的结合在 eNOS mRNA 稳定性的调节中发挥着关键作用。然而，这些蛋白质的身份仍有待确定。通过使用 RNA 亲和纯化和质谱蛋白质测序，我们发现翻译延伸因子 1 α (eEF1A) 和聚嘧啶束结合蛋白 (PTB) 特异性结合 eNOS 3'-UTR，可能调节人内皮细胞中 eNOS mRNA 的稳定性。本提案旨在继续深入探索PTB和eEF1A1在细胞培养和体内调节eNOS mRNA稳定性的机制。目标 1 将表征 eNOS mRNA 3'-UTR 与 PTB 的物理和功能相互作用，并通过 RNA 凝胶迁移率变化测定和 UV 交联测定鉴定它们的相互作用域。目标 2 将研究调节 eNOS mRNA 3'-UTR 核糖核蛋白复合物形成的分子机制。重点将放在 Rho/ROCK 通路和蛋白质-蛋白质相互作用在调节 eNOS 3'-UTR 核糖核蛋白复合物形成和 eNOS mRNA 稳定性中的作用。此外，目标 3 将研究破坏 eNOS 3'-UTR/eEF1A1/PTB 复合物是否会阻止内皮细胞中 TNF-1 诱导的 eNOS mRNA 不稳定，以及 eEF1A1 和 PTB 是否参与高胆固醇血症兔模型中内皮功能障碍的发生。这些拟议的研究将为生理和病理条件下 eNOS 表达的调节机制提供更深入的见解。希望从拟议的研究中获得的结果将为治疗心血管疾病带来新的治疗靶点。