The overall goal of this revised renewal application is to elucidate the oxidative, nitrative, and nitrosative reactions initiated by proinflammatory reactive nitrogen species (RNS) and reactive oxygen species (ROS) in cellular and biological systems. Research during the previous funding period highlighted the importance of long-range electron transfer in influencing tyrosyl nitration and cysteine oxidation in model peptides. Hypothesis: We hypothesize that through-space or through-bond electron transfer reactions induced by ROS/RNS (ONOO-, MPO/H2O2/NO2-, and NO/O2) largely mediate the nitrosation, nitration, and oxidation of peptidyl and protein cysteine, tyrosine and methionine residues.
Specific aims : First, we will determine the effects of intramolecular electron transfer reaction on tyrosyl nitration and cysteine nitrosation and oxidation of model cysteinyltyrosyl (e.g., YC) and methionyltyrosyl (e.g., YM) peptides. As an extension of this aim, we will investigate the intramolecular electron transfer mechanism from tyrosyl radical to cysteinyl residues in heme proteins (e.g., hemoglobin). Next, we will investigate the effect of hydrophobicity and electron-transfer mechanism on tyrosyl nitration and cysteine oxidation/nitrosation. To this end, we will compare the S-nitrosation and tyrosyl nitration of transmembrane peptides containing both cysteine and tyrosine in membranes with hydrophilic peptides containing cysteine and tyrosine. The objective here is to monitor nitrosation of cysteinyl residue and nitration of tyrosyl residue located at different positions in transmembrane peptide treated with RNS (e.g., peroxynitrite, NO/O2). Finally, we will assess the antiapoptotic and antinitration potentials of YC/YM peptides and mitochondria-targeted peptides in macrophage cells treated with inflammatory agents (e.g., lipopolysaccharide). Methods: Novel transmembrane peptides, cell-permeable and mitochondria-targeted cysteinyltyrosyl peptides will be synthesized and purified, and their nitration/nitrosation/oxidation products and intermediates analyzed by various techiques. We will use HPLC/UV or fluorescence, HPLC/MS, EPR and immuno-spin-trapping, stopped-flow UV/Vis, and pulse-radiolysis techniques. Significance: Nitrated, nitrosated, and oxidized lipids and proteins have been isolated in several cardiovascular, pulmonary, and neurodegenerative diseases. These products presumably act as mediators of the pathological development of inflammatory diseases. Development of targeted antinitration therapeutics is emerging as an attaractive strategy to combat these toxicities. Novelty: This comprehensive analysis of RNS reactions, using well-defined model peptides, mitochondrially-targeted peptides, and heme proteins, will provide new mechanistic insights on posttranslational nitration and nitrosation reactions induced by RNS in human disease processes.
STATEMENT: We anticipate that this comprehensive analysis of nitrative and nitrosative reactions using well-defined model peptide systems, mitochondrially-targeted peptides, and proteins will provide new mechanistic insights into post-translational modifications induced by pro-inflammatory reactive nitrogen species in human cardiovascular and neurodegnerative disease processes (e.g., atherosclerosis, diabetes, Parkinson's and Lou Gehrig's).
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