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: 7945831
• 负责人: Jianxin Sun
• 金额: $ 0.25万
• 依托单位: UNIV OF MED/DENT OF NJ-NJ MEDICAL SCHOOL
• 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 2010-11-09
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7945831
• 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. PUBLIC HEALTH RELEVANCE: The proposed studies will reveal fundamental principles that govern endothelial nitric oxide synthase (eNOS) mRNA decay and protein expression in endothelial cells. Since vascular dysregulation of eNOS expression occurs and contributes to the development of endothelial dysfunction associated with many human diseases (e.g., hypertension, coronary artery disease, chronic heart failure, peripheral artery disease, diabetes, and chronic renal failure, etc.), the proposed study may well provide significant insights into the molecular mechanisms underlying these conditions and thus facilitate the development of novel therapeutic agents.

描述（由申请人提供）：由内皮一氧化氮合酶（ENOS）产生的一氧化氮（NO），对于通过促进血管舒张和抑制血管平滑肌细胞的增殖，血小板聚集和白细胞粘合剂来维持血管稳态至关重要。尽管最初认为ENOS是本构的，但后来证明了几种病理生理刺激，例如剪切应力，慢性运动和亚综合生长态，上调eNOS表达。相反，包括肿瘤坏死因子-Alpha，缺氧和高浓度的氧化LDL降低eNOS水平的细胞因子可能有助于与许多与许多心血管疾病有关的内皮功能障碍，例如动脉粥样硬化和心脏衰竭。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。积累的证据表明，胞质蛋白与eNOS 3'未翻译区（3'-UTR）的结合在调节eNOS mRNA稳定性中起关键作用。但是，这些蛋白质的身份仍有待确定。通过使用质谱法使用RNA亲和力纯化和蛋白质测序，我们揭示了翻译延伸因子-1α（EEF1A）和多吡啶丁胺段结合蛋白（PTB）特异性结合了eNOS 3'-UTR，潜在调节人内皮细胞中潜在调节的enos mRNA稳定性。该建议将继续深入探索PTB和EEF1A1调节细胞培养和体内ENOS mRNA稳定性的机制。 AIM 1将表征eNOS mRNA 3'-UTR与PTB的物理和功能相互作用，并通过RNA凝胶迁移率转移测定法和UV-Cross链接测定法识别其相互作用域。 AIM 2将研究调节eNOS mRNA 3'-UTR核糖核蛋白复合物的分子机制。重点将放在Rho/Rock途径和蛋白质蛋白相互作用中的作用上，并在ENOS 3'-UTR核糖核蛋白复合蛋白复合物和eNOS mRNA稳定性的调节中。此外，AIM 3将调查ENOS 3'-UTR/EEF1A1/PTB复合物的破坏是否会防止TNF-1在内皮细胞中诱导的ENOS mRNA不稳定，以及EEF1A1和PTB是否参与EEF1A1和PTB是否参与内皮功能障碍的发育。这些提出的研究将提供更多关于在生理和病理条件下如何调节eNOS表达的机制的信息。希望从拟议的研究中获得的结果将导致用于治疗心血管疾病的新型治疗靶点。 公共卫生相关性：拟议的研究将揭示控制内皮细胞中内皮一氧化氮合酶（ENOS）mRNA衰变和蛋白质表达的基本原理。由于eNOS表达的血管失调发生并有助于内皮功能障碍的发展与许多人类疾病相关（例如，高血压，冠状动脉疾病，慢性心脏衰竭，慢性心力衰竭，周围动脉疾病，糖尿病和慢性肾功能衰竭等），因此拟议的研究很可能会促进这些疾病的发展。代理商。