Diabetic Vasculopathy and Mitochondrial eNOS

糖尿病血管病变和线粒体 eNOS

• 批准号: 7350221
• 负责人: Steven S Gross
• 金额: $ 42万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-02-01 至 2012-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7350221
• 关键词: 7,8-dihydrobiopterin; Affinity; Amputation; Animal Model; Anti-Inflammatory Agents; Anti-inflammatory; Basic Science; Binding; Biopterin; Blindness; Blood Vessels; Bos taurus; Cattle; Cell Line; Cessation of life; Chronic; Complications of Diabetes Mellitus; Condition; Development; Diabetes Mellitus; Diffusion; Electron Transport; Endopeptidase K; Endothelial Cells; Engineering; Equilibrium; Exhibits; Face; Functional disorder; Generations; Genetic Models; Glucose; Goals; Golgi Apparatus; Health; Hyperglycemia; Kidney Diseases; Kidney Failure; Lesion; Measurement; Mediating; Membrane; Mitochondria; Modeling; Modification; Molecular; Mus; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Oxidants; Parents; Pathogenesis; Peptides; Peroxonitrite; Pharmacotherapy; Physiological; Placement; Play; Positioning Attribute; Production; Protein Isoforms; Proteins; Proteomics; Ramp; Rate; Reaction; Regulation; Relative (related person); Renal function; Reporting; Research; Role; SKIL gene; Signal Transduction; Site; Source; Stress; Superoxides; Testing; Tissues; Translations; Vascular Endothelial Cell; base; blood pressure regulation; cofactor; diabetes mellitus therapy; diabetic; feeding; human NOS3 protein; novel; oxidation; protein aminoacid sequence; protein protein interaction; research study; restoration; tetrahydrobiopterin; type I and type II diabetes

DESCRIPTION (provided by applicant): Nitric oxide (NO) is produced by endothelial NO synthase (eNOS) and plays a key role in maintaining vascular health and renal function. Diabetic levels of glucose promote oxidation of tetrahydrobiopterin (BH4), an essential eNOS cofactor, resulting in accumulation of dihydrobiopterin (BH2). BH4 insufficiency triggers a switch in the eNOS product from NO to superoxide, resulting in endothelial dysfunction (ED), a major diabetic complication that leads to blindness, amputations, kidney failure and death. We discovered that BH4 and BH2 exhibit equal binding affinity for eNOS and infer that the balance of these species is a major determinant of vascular health. Mitochondria (Mt) are hypothesized to provide the source of superoxide that initiates BH4 oxidation in diabetes, whereas BH2-bound (uncoupled) eNOS derived superoxide may sustain BH4 oxidation and cause ED. Notably, we showed that eNOS directly associates with Mt via a pentabasic peptide in the autoinhibitory domain of eNOS (residues 629-633 in the bovine isoform) and a proteinase K-cleavable site on the outer Mt membrane. We hypothesize that this protein- protein interaction is dynamic and contributes to the NO-mediated regulation of Mt activities. Localization at the outer membrane strategically places eNOS in proximity to the major source of cellular superoxide, emanating from the Mt inner membrane due to inefficiencies in electron transport. Owing to the diffusion- limited reaction of eNOS-derived NO with electron transport-derived superoxide, a gradient of peroxynitrite would arise at the interface of these two fluxes, at the intermembrane space in Mt. Notably, the rate of electron transport-generated superoxide is accelerated by hyperglycemia - accordingly, we hypothesize that in diabetic blood vessels peroxynitrite production by Mt would accelerate, increasing the oxidation of BH4, leading to superoxide-producing, BH2-bound, eNOS on Mt. Redistribution of this uncoupled eNOS from Mt to other subcellular loci would promote BH4 oxidation at non-Mt sites, disseminating the NO insufficiency. Aim 1 of this research is to define the molecular basis for eNOS association with Mt, the consequences for NO production by eNOS and targets of eNOS-derived NO in Mt. Studies will rely on our development of strategies for the selective placement and displacement of Mt eNOS. We will employ engineered cell lines and a novel proteomic approach for unbiased identification of proteins and their specific Cys residues that undergo reversible S-nitrosylation. Preliminary experiments have already identified endogenous SNO- modified proteins in mitochondria from NOS-rich tissues - the functional consequences of these modifications remain to be established. Aim 2 will test the hypothesis that mitochondria are the primary site of glucose and oxLDL-induced BH4 oxidation, resulting in suppressed NO signaling. Aim 3 will evaluate N?- hydroxyarginine as a superoxide-dependent NO donor, for its ability to protect against BH4 oxidation, vascular lesion development and endothelial dysfunction in a murine genetic model of diabetes. This aim is a direct translation of our basic research and may provide for the selective delivery of NO to vascular sites where superoxide overproduction is greatest and hence. NO bioactivity is most compromised.

描述（由申请人提供）：一氧化氮（NO）由内皮NO合酶（eNOS）产生，在维持血管健康和肾功能中起关键作用。糖尿病水平的葡萄糖促进四氢生物蝶呤（BH 4）的氧化，一种必需的eNOS辅因子，导致二氢生物蝶呤（BH 2）的积累。BH 4不足触发eNOS产物从NO转换为超氧化物，导致内皮功能障碍（艾德），这是一种主要的糖尿病并发症，导致失明、截肢、肾衰竭和死亡。我们发现BH 4和BH 2对eNOS表现出相同的结合亲和力，并推断这些物种的平衡是血管健康的主要决定因素。线粒体（Mt）被假设为提供引发糖尿病中BH 4氧化的超氧化物的来源，而BH 2结合的线粒体（Mt）被假设为提供引发糖尿病中BH 4氧化的超氧化物的来源。（未偶联的）eNOS衍生的超氧化物可以维持BH 4氧化并引起ED。值得注意的是，我们发现eNOS通过eNOS自身抑制结构域中的五元肽直接与MT结合（牛同种型中的残基629-633）和外Mt膜上的蛋白酶K可切割位点。我们推测，这种蛋白质-蛋白质相互作用是动态的，有助于NO介导的调节MT活动。在外膜的定位战略性地将eNOS放置在细胞超氧化物的主要来源附近，由于电子传递效率低下而从Mt内膜发出。由于eNOS衍生的NO与电子传递衍生的超氧化物的扩散限制反应，在这两种通量的界面处，在Mt.值得注意的是，电子传递产生的超氧化物的速率被高血糖症加速-因此，我们假设在糖尿病血管中由Mt产生的过氧亚硝酸盐将加速，增加BH 4的氧化，导致Mt上产生超氧化物、BH 2结合的eNOS。重新分配这种解偶联eNOS从MT到其他亚细胞位点将促进BH 4氧化在非MT网站，传播NO不足。本研究的目的1是确定eNOS与Mt相关的分子基础，eNOS产生NO的后果以及Mt中eNOS衍生的NO的靶点。研究将依赖于我们的战略的选择性安置和位移的eNOS山的发展。我们将采用工程细胞系和一种新的蛋白质组学方法进行无偏鉴定的蛋白质和其特定的半胱氨酸残基，进行可逆的S-亚硝基化。初步的实验已经在富含NOS的组织的线粒体中鉴定出内源性SNO修饰的蛋白质-这些修饰的功能结果仍有待确定。目的2将检验线粒体是葡萄糖和oxLDL诱导的BH 4氧化的主要位点，导致抑制NO信号传导的假设。目标3将评估N？-羟基精氨酸作为超氧化物依赖性NO供体，在糖尿病的鼠遗传模型中保护免受BH 4氧化、血管损伤发展和内皮功能障碍的能力。这一目标是我们的基础研究的直接翻译，并可能提供选择性地将NO输送到超氧化物过量产生最大的血管部位，因此。没有生物活性受到最大损害。