Reactive nitrogen species and accelerated atherosclerosis in type I diabetes

描述(申请人提供)：在上一次资助期间，我们发现活性氮物种如过氧亚硝酸盐(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)}，糖尿病患者eNOS解偶联会加速动脉粥样硬化。此外，我们还发现四氢生物蝶呤(BH4)缺乏是内皮型一氧化氮合酶(ENOS)解偶联的关键。最后，我们报告了BH4缺乏是由于内皮细胞中泛素-蛋白酶体系统(UPS)迅速降解了BH4从头合成中的限速酶GTP环水解酶I(GTPCH1；E.C.3.5.4.16)。然而，为什么GTPCH1会受到糖尿病的影响还没有得到解决。因此，该项目将检验这样的假设，即GTPCH1锌结合结构的氧化会使酶失活，导致BH4缺乏症，从而导致糖尿病患者eNOS解偶联。目的1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-组氨酸络合物的氧化破坏是否促进泛素化和随之而来的蛋白酶体降解。目的2探讨高血糖抑制内皮细胞GTPCH1的分子机制。目的3确定ONOO诱导的GTPCH1抑制和泛素化在糖尿病增强的动脉粥样硬化小鼠体内模型中的作用。我们相信，这些拟议的研究将提供新的信息，了解与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的影响，以及清除ONOO-是否是治疗糖尿病的有效方法。

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.