Diabetic Vasculopathy and Mitochondrial eNOS

糖尿病血管病变和线粒体 eNOS

基本信息

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
项目类别：
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

项目摘要

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，并在维持血管健康和肾功能中起关键作用。葡萄糖的糖尿病水平促进四氢无菌蛋白（BH4）的氧化，这是一种必不可少的eNOS辅因子，导致二氢蛋白蛋白酶的积累（BH2）。 BH4功能不全会触发eNOS产品从NO到超氧化物的开关，从而导致内皮功能障碍（ED），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BH4和BH2对eNOS具有相同的结合亲和力，并推断出这些物种的平衡是血管健康的主要决定因素。假设线粒体（MT）提供了糖尿病中BH4氧化的超氧化物的来源，而BH2结合（未偶联的）eNOS衍生的超氧化物可能维持BH4氧化并导致ED。值得注意的是，我们表明eNOS通过eNOS自身抑制域（牛同工型中的残基629-633）和蛋白酶K可辨认位点的eNOS与MT直接相关。我们假设这种蛋白质蛋白质相互作用是动态的，并有助于MT活性的无介导的调节。由于电子传输的效率低下而引起的，在远离主要细胞超氧化物的主要来源的eNOS上，在外膜上进行定位。由于eNOS衍生的NO与电子传输源性超氧化物的扩散有限反应有限，因此在这两个通量的界面上，在山上的膜间空间处出现过氧亚硝酸盐的梯度。通过MT会加速，增加BH4的氧化，从而导致产生超氧化物的BH2结合，MT上的eNOS。将这种未偶联的ENOS从MT重新分布从MT重新分布到其他亚细胞基因座，将促进非MT位点的BH4氧化，从而在非MT部位促进无毫无用处。这项研究的目标1是定义eNOS与MT关联的分子基础，eNOS和eNOS衍生的NO的NO生产的后果将依赖于我们制定的策略来选择性放置和位移MT Enos。我们将采用工程的细胞系和一种新型的蛋白质组学方法，以无偏鉴定蛋白质及其特定的CYS残基，这些残基经历了可逆的S-硝基化。初步实验已经从富含NOS的组织中鉴定出线粒体中的内源性SNO蛋白 - 这些修饰的功能后果仍有待确定。 AIM 2将检验以下假设：线粒体是葡萄糖和OXLDL诱导的BH4氧化的主要部位，导致未抑制没有信号传导。 AIM 3将评估N？ - 羟基氨基氨酸作为超氧化物依赖性的NO供体，因为它可以防止BH4氧化，血管病变发育和内皮功能障碍在糖尿病的鼠类遗传模型中。这个目的是我们基础研究的直接翻译，可以选择为超氧化物过量生产最大的血管部位选择性传递。没有生物活性最大。