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

• 批准号: 7893780
• 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
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-30 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7893780
• 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; Cells; Coupled; Cultured Cells; Cysteine; Data; Development; Diabetes Mellitus; Diabetic mouse; Disease; 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; 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; Proteins; Reactive Nitrogen Species; Reactive Oxygen Species; Recombinants; Role; Source; Streptozocin; 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; overexpression; oxidant stress; oxidation; polyethylene glycol-superoxide dismutase; protein protein interaction; tetrahydrobiopterin; treatment strategy; type I and type II diabetes; zinc tetrathiolate cluster

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负责高血糖症（I型和II型糖尿病的主要特征）引起的内皮型一氧化氮合酶（eNOS）解偶联的分子机制。为了实现这一目标，我们首先提出了一个详细的检查氧化应激和eNOS活性的内皮细胞由于高血糖症。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.-）的机制。代替一氧化氮（NO），利用表明高血糖症通过过氧亚硝酸盐（ONOO-）激活26 S蛋白酶体以通过增加鸟苷三磷酸环化水解酶I（GTP-CH，EC3.5.4.16）（BH 4合成的限速酶）的降解来降低四氢生物蝶呤（BH 4）水平的初步数据。由高血糖症驱动的氧化应激同时氧化eNOS的四硫醇锌（ZnS 4）中心，这是NO产生所需的一种形式。有了这些信息，我们将研究如何高血糖症或ONOO-激活蛋白酶体和26 S蛋白酶体亚基的氧化修饰的特点，通过使用串联质谱结合肽指纹图谱。在我们申请的第二部分中，我们将开发一种锌缺陷型eNOS突变体（eNOS-C94 A），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，用于在COS-7和内皮细胞中表达，以研究其对NO生物活性和氧化剂产生的影响。此外，我们建议繁殖过表达eNOS-C94 A突变体的转基因小鼠，我们将尝试将这种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-C94 A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对糖尿病增强的动脉粥样硬化病变形成的影响。这些研究将为糖尿病相关的代谢应激如何导致内皮损伤提供新的信息。