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.
Published data and preliminary data included in this competitive renewal application demonstrate that eNOS uncoupling causes accelerated atherosclerosis in type 1 diabetes. How diabetes uncouples eNOS is unknown. GTPCH1 is the rate-limiting enzyme for the synthesis of tetrahydrobiopterin, an essential co-factor for eNOS. The goal of this competitive renewal application is to determine 1) how diabetes inhibits GTPCH1; and 2) to determine the contribution of GTPCH1 inhibition in diabetes-enhanced atherosclerosis in mouse models of atherosclerosis in vivo.
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