Mitochondria in eNOS Function and Dysfunction

我们将在过去资金时期的发现基础上，阐明线粒体(Mt)的作用。 内皮型一氧化氮合酶的功能和功能障碍。而腔泡膜和高尔基膜被认为是主要的 ENOS在内皮细胞中的表达部位，我们发现eNOS也通过五基结合 其自身抑制区(牛eNOS残基629-633)的多肽序列与一种蛋白水解酶K- 山体外膜上的可裂解部位。我们假设eNOS的这种蛋白质-蛋白质相互作用是 并参与NO对线粒体活性的调节。与外膜结合 的战略定位eNOS接近细胞超氧化物的主要来源，源自 MT内膜由于电子传输效率低下。由于……的扩散受限反应 ENOS衍生的NO与电子传递衍生的超氧化物，过氧亚硝酸盐的梯度将出现在 这两种通量的界面，从线粒体膜间间隙发出。在环境中 疾病相关的氧化应激(例如，暴露于高血糖或氧化低密度脂蛋白)，我们假设 Mt产生过氧亚硝酸盐会加速，增加BH4的氧化，导致BH2结合 未耦合的enos在Mt.随后将去偶联的mt eNOS重新分布到其他亚细胞基因座 促进BH4在非线粒体部位的氧化，传播没有不足。目标1是定义 内皮型一氧化氮合酶与线粒体结合的分子基础、调控线粒体eNOS活性的机制及鉴定 内皮型一氧化氮合酶在山区的靶标。研究将依赖于我们为选择性地制定战略 Mt enos的放置和位移。我们将使用工程细胞系和一种新的蛋白质组 可逆S亚硝化后蛋白质和半胱氨酸残基的无偏鉴别方法。 初步实验已经在线粒体中鉴定出内源性SNO修饰的蛋白质 富含一氧化氮合酶的组织--这些修饰的功能后果仍有待确定。目标2将 测试线粒体是葡萄糖和oxLDL诱导的BH4氧化的主要部位的假设， 导致被抑制的无信号。AIM 3将评估NG-羟基精氨酸是一种超氧化物依赖 NO供体，因为它有能力防止BH4氧化、血管病变和内皮细胞的形成 动脉粥样硬化形成的小鼠模型的功能障碍。这个目标是对所进行的基础研究的直接翻译 在前一个资助期内--侧重于选择性输送NOTO血管部位的研究 超氧化物的过量生产是最大的，因此，没有生物活性是最有限的。

We will build on discoveries made during the past funding period by elucidating the role of mitochondria(Mt) in eNOS function and dysfunction. While caveolar and golgi membranes are recognized as the predominant sites of eNOS expression in endothelial cells, we discovered that eNOS also associates via a pentabasic peptide sequence in its autoinhibitory domain (bovine eNOS residues 629-633) with a proteinase K- cleavable site on the outer membrane of Mt. We hypothesize that this protein-protein interaction of eNOS is dynamic and contributes to the regulation of mitochondrial activities by NO. Binding to the outer membrane of Mt strategically positions eNOS in proximity to the major source of cellular superoxide, orginating 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, emanating from the mitochondrial inter-membrane space. In the setting of disease-associated oxidant stresses (e.g., exposure to elevated glucose or oxidized LDL), we hypothesize that peroxynitrite production by Mt would accelerate, increasing the oxidation of BH4, leading BH2-bound uncoupled eNOS on Mt. Subsequent redistribution of uncoupled Mt eNOS to other subcellular loci would promote BH4 oxidation at non-mitochondrial sites, disseminating NO insufficiency. Aim 1 is to define the molecular basis for eNOS association with Mt, mechanisms that regulate eNOS activity at Mt and identify 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 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 NG-hydroxyarginine as a superoxide-dependent NO donor, for its ability to protect against BH4 oxidation, vascular lesion development and endothelial dysfunction in a murine model of atherogenesis. This aim is a direct translation of basic research performed during the prior funding period - studies which focus on the selective delivery of NOto vascular sites where superoxide overproduction is greatest and hence, NO bioactivity is most limited.