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 predominantsites of eNOS expression in endothelial cells, we discovered that eNOS also associates via a pentabasicpeptide 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 isdynamic and contributes to the regulation of mitochondrial activities by NO. Binding to the outer membraneof Mt strategically positions eNOS in proximity to the major source of cellular superoxide, orginating from theMt inner membrane due to inefficiencies in electron transport. Owing to the diffusion-limited reaction ofeNOS-derived NO with electron transport-derived superoxide, a gradient of peroxynitrite would arise at theinterface of these two fluxes, emanating from the mitochondrial inter-membrane space. In the setting ofdisease-associated oxidant stresses (e.g., exposure to elevated glucose or oxidized LDL), we hypothesizethat peroxynitrite production by Mt would accelerate, increasing the oxidation of BH4, leading BH2-bounduncoupled eNOS on Mt. Subsequent redistribution of uncoupled Mt eNOS to other subcellular loci wouldpromote BH4 oxidation at non-mitochondrial sites, disseminating NO insufficiency.
Aim 1 is to define themolecular basis for eNOS association with Mt, mechanisms that regulate eNOS activity at Mt and identifytargets of eNOS-derived NO in Mt. Studies will rely on our development of strategies for the selectiveplacement and displacement of Mt eNOS. We will employ engineered cell lines and a novel proteomicapproach for unbiased identification of proteins and Cys residues that undergo reversible S-nitrosylation.Preliminary experiments have already identified endogenous SNO-modified proteins in mitochondria fromNOS-rich tissues - the functional consequences of these modifications remain to be established.
Aim 2 willtest 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-dependentNO donor, for its ability to protect against BH4 oxidation, vascular lesion development and endothelialdysfunction in a murine model of atherogenesis.
This aim i s a direct translation of basic research performedduring the prior funding period - studies which focus on the selective delivery of NOto vascular sites wheresuperoxide overproduction is greatest and hence, NO bioactivity is most limited.
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