Reactive nitrogen species and accelerated atherosclerosis in type I diabetes

描述（由申请人提供）：在上一个资助期间，我们发现活性氮物质，如过氧亚硝酸盐（ONOO-）解偶联内皮型一氧化氮合酶（eNOS）{产生超氧阴离子（O2.-）或ONOO-而不是一氧化氮（NO）}，并且糖尿病中eNOS解偶联导致动脉粥样硬化加速。此外，我们发现，四氢生物蝶呤（BH 4）缺乏，eNOS的一个重要的辅因子，是在糖尿病eNOS解偶联的发展的关键。最后，我们报告说，BH 4缺乏是由于快速降解的GTP环化水解酶I（GTPCH 1; E.C. 3.5.4.16），BH 4从头合成中的限速酶，通过内皮细胞中的泛素-蛋白酶体系统（UPS）。然而，为什么GTPCH 1会受到糖尿病的影响还没有得到解决。因此，该项目将测试以下假设：GTPCH 1的锌结合结构的氧化使酶失活，导致BH 4缺乏，从而导致糖尿病中eNOS解偶联。目的1是确定锌在维持GTPCH 1活性和稳定性中的重要作用，以及GTPCH 1中锌-半胱氨酸-组氨酸复合物的氧化破坏是否增强泛素化和随后的蛋白酶体降解。目的2探讨高血糖抑制内皮细胞GTPCH 1表达的分子机制。目的3是确定ONOO-诱导的GTPCH 1抑制和泛素化在糖尿病增强的小鼠动脉粥样硬化模型中的作用。我们相信，拟议的研究将提供新的信息，如何与糖尿病相关的代谢应激导致内皮细胞损伤，内皮细胞如何试图保护自己免受这些压力，以及清除ONOO-是否是糖尿病的有效疗法。

DESCRIPTION (provided by applicant): During last funding period, we have found that reactive nitrogen species such as peroxynitrite (ONOO-) uncouples endothelial nitric oxide synthase (eNOS) {generates superoxide anions (O2.-) or ONOO- instead of nitric oxide (NO)} and that eNOS uncoupling in diabetes causes accelerated atherosclerosis. Further, we found that tetrahydrobiopterin (BH4) deficiency, an essential cofactor for eNOS, is the key in the development of eNOS uncoupling in diabetes. Finally, we report that BH4 deficiency is due to rapid degradation of GTP cyclohydrolase I (GTPCH1; E.C. 3.5.4.16), the rate-limiting enzyme in BH4 de novo synthesis, by ubiquitin-proteasome system (UPS) in endothelial cells. However, why GTPCH1 is affected by diabetes hasn't been addressed. Thus, this project will test the hypothesis that oxidation of the zinc-binding structures of GTPCH1 inactivates the enzyme resulting in BH4 deficiency with consequent eNOS uncoupling in diabetes. Aim 1 is establish the essential role of zinc in maintaining GTPCH1 activity and stability and if oxidative disruption of the zinc-cysteine- histidine complexation in GTPCH1 enhances ubiquitination and consequent proteasomal degradation. Aim 2 is to investigate the molecular mechanisms by which hyperglycemia inhibits GTPCH1 in endothelial cells. Aim 3 is to determine the contributions of ONOO--induced GTPCH1 inhibition and ubiquitination in diabetes-enhanced atherosclerosis in mouse models of atherosclerosis in vivo. We believe that the proposed studies will provide novel information as to how the metabolic stress associated with diabetes causes damage to the endothelium and how the endothelial cell attempts to protect itself against these stresses and whether scavenging ONOO- is an effective therapy for diabetes.