We will build on discoveries made during the past funding period by elucidating the role of mitochondria(Mt) in eNOS function and dysfunction. While caveolar and golgi membranes are recognized as the predominant sites of eNOS expression in endothelial cells, we discovered that eNOS also associates via a pentabasic peptide sequence in its autoinhibitory domain (bovine eNOS residues 629-633) with a proteinase K- cleavable site on the outer membrane of Mt. We hypothesize that this protein-protein interaction of eNOS is dynamic and contributes to the regulation of mitochondrial activities by NO. Binding to the outer membrane of Mt strategically positions eNOS in proximity to the major source of cellular superoxide, orginating from the Mt inner membrane due to inefficiencies in electron transport. Owing to the diffusion-limited reaction of eNOS-derived NO with electron transport-derived superoxide, a gradient of peroxynitrite would arise at the interface of these two fluxes, emanating from the mitochondrial inter-membrane space. In the setting of disease-associated oxidant stresses (e.g., exposure to elevated glucose or oxidized LDL), we hypothesize that peroxynitrite production by Mt would accelerate, increasing the oxidation of BH4, leading BH2-bound uncoupled eNOS on Mt. Subsequent redistribution of uncoupled Mt eNOS to other subcellular loci would promote BH4 oxidation at non-mitochondrial sites, disseminating NO insufficiency.
Aim 1 is to define the molecular basis for eNOS association with Mt, mechanisms that regulate eNOS activity at Mt and identify targets of eNOS-derived NO in Mt. Studies will rely on our development of strategies for the selective placement and displacement of Mt eNOS. We will employ engineered cell lines and a novel proteomic approach for unbiased identification of proteins and Cys residues that undergo reversible S-nitrosylation. Preliminary experiments have already identified endogenous SNO-modified proteins in mitochondria from NOS-rich tissues - the functional consequences of these modifications remain to be established.
Aim 2 will test the hypothesis that mitochondria are the primary site of glucose and oxLDL-induced BH4 oxidation, resulting in suppressed NO signaling.
Aim 3 will evaluate NG-hydroxyarginine as a superoxide-dependent NO donor, for its ability to protect against BH4 oxidation, vascular lesion development and endothelial dysfunction in a murine model of atherogenesis.
This aim i s a direct translation of basic research performed during the prior funding period - studies which focus on the selective delivery of NOto vascular sites where superoxide overproduction is greatest and hence, NO bioactivity is most limited.
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