Diabetic Vasculopathy and Mitochondrial eNOS

描述（由申请人提供）：一氧化氮（NO）是由内皮NO合成酶（eNOS）产生的，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，这是一种必要的eNOS辅助因子，导致双氢生物蝶呤（BH2）的积累。BH4不足触发eNOS产物从NO到超氧化物的转换，导致内皮功能障碍（ED），这是一种主要的糖尿病并发症，可导致失明、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）被认为是糖尿病中启动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，我们发现eNOS通过eNOS自抑制区域的五基肽（牛同型中的残基629-633）和Mt外膜上的蛋白酶k可切割位点与Mt直接结合。我们假设这种蛋白-蛋白相互作用是动态的，并有助于no介导的Mt活性调节。外膜定位战略性地将eNOS置于细胞超氧化物的主要来源附近，由于电子传递效率低下，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，在Mt的膜间空间，会产生一个过氧亚硝酸盐的梯度。值得注意的是，电子传递产生的超氧化物的速率被高血糖加速-因此，我们假设在糖尿病血管中，Mt会加速过氧亚硝酸盐的产生，增加BH4的氧化，导致超氧化物的产生，bh2结合。Mt上的eNOS。这种未偶联的eNOS从Mt重新分布到其他亚细胞位点会促进非Mt位点的BH4氧化，传播NO不足。本研究的目的1是确定eNOS与Mt关联的分子基础，eNOS产生NO的后果以及eNOS衍生的NO在Mt中的靶标。研究将依赖于我们对Mt eNOS选择性放置和置换策略的开发。我们将采用工程细胞系和一种新的蛋白质组学方法来无偏鉴定蛋白质及其特定的经过可逆s -亚硝基化的Cys残基。初步实验已经在富含nos的组织的线粒体中发现了内源性SNO修饰蛋白，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氧化低密度脂蛋白诱导的BH4氧化的主要部位，导致NO信号被抑制的假设。目标3将计算N？-羟精氨酸作为一种超氧化物依赖性NO供体，在小鼠糖尿病遗传模型中具有抗BH4氧化、血管病变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，并可能提供选择性输送NO到血管部位，超氧化物过量生产是最大的，因此。NO的生物活性受损最严重。

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.