Reactive Nitrogen and Accelerated Atherosclerosis in Type I Diabetes

活性氮与 I 型糖尿病加速动脉粥样硬化

基本信息

批准号：
7373939
负责人：
MING-HUI ZOU
金额：
$ 36.63万
依托单位：
UNIVERSITY OF OKLAHOMA HLTH SCIENCES CTR
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-09-30 至 2013-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7373939
关键词：
26S proteasome 3-nitrotyrosine Abbreviations Acute Affect Alanine Animal Model Aorta Apolipoprotein E Arterial Fatty Streak Atherosclerosis Binding Sites Biopterin Blood Vessels Breeding Cardiovascular Diseases Cardiovascular Models Cells Condition Coupled Cultured Cells Cysteine Data Development Diabetes Mellitus Diabetic mouse Disease Disruption Dissociation Endothelial Cells Endothelium Enzyme Uncoupling Enzymes Exhibits GTP Cyclohydrolase I Generations Glucose Goals Guanosine Triphosphate Heart Heart Hypertrophy Human Hyperglycemia Hypertension Injury Insulin-Dependent Diabetes Mellitus Kidney Knock-out L-Glucose Link Low Density Lipoprotein Receptor Low-Density Lipoproteins MG132 Maintenance Mass Spectrum Analysis Metabolic stress Mitochondria Modification Molecular Mus Mutation NAD(P)H oxidase NG-Nitroarginine Methyl Ester Nitric Oxide Nitrogen Oxidants Oxidases Oxidative Stress Oxygen Patients Peptide Mapping Peroxonitrite Phosphorylation Polyethylene Glycols Post-Translational Protein Processing Production Prostacyclin synthase Proteasome Inhibitor Protein Kinase C Protein Overexpression Proteins Rate Reactive Nitrogen Species Reactive Oxygen Species Recombinants Role Source Streptozocin Stress Sulfhydryl Compounds Superoxide Dismutase Superoxides System Testing Transgenic Mice Transgenic Organisms Tyrosine Ubiquitination Vascular Diseases Vascular Endothelium Zinc arginine methyl ester cardiovascular risk factor clinically relevant cofactor diabetic dimer exposed human population gain of function human NOS3 protein hypercholesterolemia in vitro Model in vivo inhibitor/antagonist insight loss of function monomer mouse model multicatalytic endopeptidase complex mutant nitration novel oxidation polyethylene glycol-superoxide dismutase protein protein interaction tetrahydrobiopterin type I and type II diabetes zinc tetrathiolate cluster

项目摘要

DESCRIPTION (provided by applicant): The overall objective of this competitive renewal application is to determine the molecular mechanisms responsible for endothelial nitric oxide synthase (eNOS) uncoupling caused by hyperglycemia, a principal feature of both type I and type II diabetes. In order to achieve this objective, we first have proposed a detailed examination of oxidant stress and eNOS activity in the endothelial cell due to hyperglycemia. The next part of our proposal will determine the mechanism(s) by which this oxidant stress causes eNOS to produce superoxide anions (O2.-) instead of nitric oxide (NO), capitalizing on preliminary data indicating that hyperglycemia activates 26S proteasomes via peroxynitrite (ONOO-) to reduce levels of tetrahydrobiopterin (BH4) by increasing the degradation of guanosine triphosphate cyclohydrolase I (GTP-CH, EC3.5.4.16), the rate-limiting enzyme for BH4 synthesis. Oxidant stress driven by hyperglycemia concurrently oxidizes the zinc-tetrathiolate (ZnS4) center of eNOS, a form required for NO production. With this information, we will examine how hyperglycemia or ONOO- activates proteasomes and characterize the oxidative modifications of 26S proteasome subunits by using tandem mass spectroscopy coupled with peptide fingerprinting. In the second part of our application, we will develop a zinc-deficient eNOS mutant (eNOS-C94A) in which the eNOS residue cysteine 94, the zinc-binding site within the interface of the enzyme-active eNOS dimers, is replaced by alanine, for expression in both COS-7 and endothelial cells to examine its implications for NO bioactivity and oxidant production. Furthermore, we propose to breed transgenic mice overexpressing eNOS-C94A mutants and we will attempt to link this eNOS modification with NO bioactivity and oxidant stress in mice in vivo. Finally, crossing these transgenic mice (eNOS-C94A mutant) with apolipoprotein E (Apo-E)- or LDL- KO mice will allow us to more directly assess the effect on the formation of atherosclerotic lesions that are enhanced by diabetes. These studies will provide novel information as to how the metabolic stresses associated with diabetes cause damage to the endothelium.

描述（由申请人提供）：本竞争性更新申请的总体目标是确定高血糖引起的内皮一氧化氮合酶（eNOS）解偶联的分子机制，高血糖是 I 型和 II 型糖尿病的主要特征。为了实现这一目标，我们首先提出对高血糖引起的内皮细胞氧化应激和 eNOS 活性进行详细检查。我们提案的下一部分将确定这种氧化应激导致 eNOS 产生超氧阴离子 (O2.-) 而不是一氧化氮 (NO) 的机制，利用表明高血糖通过过氧亚硝酸盐激活 26S 蛋白酶体的初步数据。 ONOO-) 通过增加三磷酸鸟苷环水解酶 I (GTP-CH, EC3.5.4.16)，BH4 合成的限速酶。高血糖驱动的氧化应激同时氧化 eNOS 的四硫醇锌 (ZnS4) 中心，这是产生 NO 所需的一种形式。有了这些信息，我们将研究高血糖或 ONOO- 如何激活蛋白酶体，并通过使用串联质谱法和肽指纹分析来表征 26S 蛋白酶体亚基的氧化修饰。在我们申请的第二部分中，我们将开发一种缺锌的 eNOS 突变体 (eNOS-C94A)，其中 eNOS 残基半胱氨酸 94（酶活性 eNOS 二聚体界面内的锌结合位点）被丙氨酸取代，在 COS-7 和内皮细胞中表达，以检查其对 NO 生物活性和氧化剂产生的影响。此外，我们建议培育过度表达 eNOS-C94A 突变体的转基因小鼠，并尝试将这种 eNOS 修饰与小鼠体内的 NO 生物活性和氧化应激联系起来。最后，将这些转基因小鼠（eNOS-C94A突变体）与载脂蛋白E（Apo-E）或LDL-KO小鼠杂交将使我们能够更直接地评估糖尿病对动脉粥样硬化病变形成的影响。这些研究将提供关于与糖尿病相关的代谢应激如何对内皮细胞造成损害的新信息。