Reactive nitrogen species and accelerated atherosclerosis in type I diabetes

描述（由申请人提供）：在上一个资助期内，我们发现活性氮如过氧亚硝酸盐（ONOO-）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的偶联导致加速动脉粥样硬化。此外，我们发现四氢生物蝶呤（BH4）缺乏是eNOS的重要辅助因子，是糖尿病eNOS解偶联发展的关键。最后，我们报道BH4缺乏是由于内皮细胞中的泛素-蛋白酶体系统（UPS）快速降解GTP环水解酶I (GTPCH1； E.C. 3.5.4.16)，这是BH4从头合成的限速酶。然而，为什么GTPCH1受糖尿病的影响还没有解决。因此，本项目将验证GTPCH1的锌结合结构氧化使酶失活导致BH4缺乏并导致糖尿病中eNOS解偶联的假设。目的1是确定锌在维持GTPCH1活性和稳定性中的重要作用，以及GTPCH1中锌-半胱氨酸-组氨酸络合的氧化破坏是否会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GTPCH1的分子机制。目的3是在体内动脉粥样硬化小鼠模型中确定ONOO诱导的GTPCH1抑制和泛素化在糖尿病增强动脉粥样硬化中的作用。我们相信，这些研究将为糖尿病相关的代谢应激如何导致内皮细胞损伤，内皮细胞如何试图保护自己免受这些应激，以及清除ONOO-是否是一种有效的糖尿病治疗方法提供新的信息。

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.