Reactive Nitrogen and Accelerated Atherosclerosis in Type I Diabetes

描述(申请人提供)：这项竞争性更新申请的总体目标是确定由高血糖引起的内皮型一氧化氮合酶(ENOS)解偶联的分子机制，高血糖是I型和II型糖尿病的主要特征。为了实现这一目标，我们首先提出了对高血糖引起的内皮细胞氧化应激和eNOS活性的详细检查。我们提案的下一部分将确定这种氧化应激导致eNOS产生超氧阴离子(O2.-)而不是一氧化氮(NO)的机制(S)，利用初步数据表明，高血糖通过过氧亚硝酸盐(ONOO-)激活26S蛋白酶体，通过增加鸟苷三磷酸环水解酶I(GTP-CH，EC3.5.4.16)的降解来降低四氢生物蝶呤(BH4)的水平。鸟苷三磷酸环水解酶I是合成BH4的限速酶。由高血糖驱动的氧化应激同时氧化eNOS的四硫代锌中心(Zns4)，这是产生NO所必需的形式。有了这些信息，我们将研究高血糖或ONOO-如何激活蛋白酶体，并利用串联质谱仪和多肽指纹图谱来表征26S蛋白酶体亚基的氧化修饰。在我们的应用的第二部分，我们将开发一个缺锌的eNOS突变体(eNOS-C94A)，其中eNOS残基半胱氨酸94(酶活性eNOS二聚体界面上的锌结合部位)被丙氨酸取代，用于在COS-7和内皮细胞中表达，以检测其对NO生物活性和氧化剂产生的影响。此外，我们建议培育高表达eNOS-C94A突变体的转基因小鼠，并试图在体内将这种eNOS修饰与小鼠体内的无生物活性和氧化应激联系起来。最后，将这些转基因小鼠(eNOS-C94A突变体)与载脂蛋白E(Apo-E)或低密度脂蛋白-KO小鼠杂交，将使我们能够更直接地评估糖尿病对动脉粥样硬化病变形成的影响。这些研究将提供与糖尿病相关的代谢应激如何导致内皮损伤的新信息。

DESCRIPTION (provided by applicant): The overall objective of this competitive renewal application is to determine the molecular mechanisms responsible for endothelial nitric oxide synthase (eNOS) uncoupling caused by hyperglycemia, a principal feature of both type I and type II diabetes. In order to achieve this objective, we first have proposed a detailed examination of oxidant stress and eNOS activity in the endothelial cell due to hyperglycemia. The next part of our proposal will determine the mechanism(s) by which this oxidant stress causes eNOS to produce superoxide anions (O2.-) instead of nitric oxide (NO), capitalizing on preliminary data indicating that hyperglycemia activates 26S proteasomes via peroxynitrite (ONOO-) to reduce levels of tetrahydrobiopterin (BH4) by increasing the degradation of guanosine triphosphate cyclohydrolase I (GTP-CH, EC3.5.4.16), the rate-limiting enzyme for BH4 synthesis. Oxidant stress driven by hyperglycemia concurrently oxidizes the zinc-tetrathiolate (ZnS4) center of eNOS, a form required for NO production. With this information, we will examine how hyperglycemia or ONOO- activates proteasomes and characterize the oxidative modifications of 26S proteasome subunits by using tandem mass spectroscopy coupled with peptide fingerprinting. In the second part of our application, we will develop a zinc-deficient eNOS mutant (eNOS-C94A) in which the eNOS residue cysteine 94, the zinc-binding site within the interface of the enzyme-active eNOS dimers, is replaced by alanine, for expression in both COS-7 and endothelial cells to examine its implications for NO bioactivity and oxidant production. Furthermore, we propose to breed transgenic mice overexpressing eNOS-C94A mutants and we will attempt to link this eNOS modification with NO bioactivity and oxidant stress in mice in vivo. Finally, crossing these transgenic mice (eNOS-C94A mutant) with apolipoprotein E (Apo-E)- or LDL- KO mice will allow us to more directly assess the effect on the formation of atherosclerotic lesions that are enhanced by diabetes. These studies will provide novel information as to how the metabolic stresses associated with diabetes cause damage to the endothelium.