Diabetic Vasculopathy and Mitochondrial eNOS

说明书(申请人提供)：一氧化氮(NO)由内皮型一氧化氮合酶(ENOS)产生，在维持血管健康和肾功能方面起着关键作用。糖尿病水平的葡萄糖促进四氢生物蝶呤(BH4)的氧化，四氢生物蝶呤(BH4)是一种基本的eNOS辅助因子，导致二氢生物蝶呤(BH2)的积累。BH4不足触发eNOS产物从NO转换为超氧化物，导致内皮功能障碍(ED)，这是糖尿病的一种主要并发症，导致失明、截肢、肾功能衰竭和死亡。我们发现BH4和BH2对eNOS表现出同等的结合亲和力，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)被认为是启动糖尿病患者BH4氧化的超氧化物的来源，而BH2结合(解偶联)的eNOS产生的超氧化物可能支持BH4的氧化并导致ED。值得注意的是，我们发现eNOS通过eNOS自身抑制区(牛的629-633残基)上的五碱基多肽和mt外膜上的蛋白酶K可裂解部位直接与mt结合。我们假设这种蛋白质-蛋白质相互作用是动态的，并有助于NO介导的线粒体活性的调节。ENOS在外膜的定位具有战略意义，使其接近细胞内超氧化物歧化的主要来源，由于电子传递效率低下，细胞内膜发出的超氧化物歧化。由于eNOS生成的NO与电子传递生成的超氧化物发生扩散受限反应，在mt.的膜间隙，这两种助熔剂的界面处会出现过亚硝酸根的梯度。值得注意的是，高血糖加速了电子传递产生超氧化物的速度--因此，我们假设在糖尿病血管中，线粒体产生过氧亚硝酸盐的速度会加快，增加BH4的氧化，导致线粒体上产生超氧化物，结合BH2，eNOS。这种未偶联的eNOS从mt重新分布到其他亚细胞位点将促进非mt位置的BH4氧化，从而传播NO不足。本研究的目的一是明确内皮型一氧化氮合酶与内皮型一氧化氮合酶结合的分子基础、内皮型一氧化氮合酶对内皮型一氧化氮合酶产生的影响以及内皮型一氧化氮合酶在内皮型一氧化氮合酶中的靶点。研究将有赖于我们制定选择性安置和置换内诺斯山的战略。我们将使用工程细胞系和一种新的蛋白质组学方法来无偏见地鉴定经历可逆S亚硝化的蛋白质及其特定的半胱氨酸残基。初步实验已经在富含一氧化氮合酶的组织的线粒体中发现了内源性SNO修饰的蛋白质-这些修饰的功能后果仍有待确定。目的2将验证线粒体是葡萄糖和oxLDL诱导的BH4氧化的主要部位的假说，导致NO信号被抑制。目的3将评估N？-羟基精氨酸作为超氧化物依赖的一氧化氮供体，在糖尿病小鼠遗传模型中预防BH4氧化、血管病变发展和内皮功能障碍的能力。这个目标是对我们的基础研究的直接翻译，并可能提供选择性地将NO输送到超氧阴离子过量最严重的血管部位，因此。任何生物活性都不会受到最大的影响。

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.