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.