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.

Public Health Relevance

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).

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Macromolecular Structure and Function A Study Section (MSFA)
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Charette, Marc F
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Medical College of Wisconsin
Schools of Medicine
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Kalyanaraman, Balaraman; Hardy, Micael; Zielonka, Jacek (2016) A Critical Review of Methodologies to Detect Reactive Oxygen and Nitrogen Species Stimulated by NADPH Oxidase Enzymes: Implications in Pesticide Toxicity. Curr Pharmacol Rep 2:193-201
Zielonka, Jacek; Sikora, Adam; Adamus, Jan et al. (2015) Detection and differentiation between peroxynitrite and hydroperoxides using mitochondria-targeted arylboronic acid. Methods Mol Biol 1264:171-81
Koto, T; Michalski, R; Zielonka, J et al. (2014) Detection and identification of oxidants formed during •NO/O2•? reaction: a multi-well plate CW-EPR spectroscopy combined with HPLC analyses. Free Radic Res 48:478-86
Zielonka, Jacek; Cheng, Gang; Zielonka, Monika et al. (2014) High-throughput assays for superoxide and hydrogen peroxide: design of a screening workflow to identify inhibitors of NADPH oxidases. J Biol Chem 289:16176-89
Michalski, Radoslaw; Zielonka, Jacek; Gapys, Ewa et al. (2014) Real-time measurements of amino acid and protein hydroperoxides using coumarin boronic acid. J Biol Chem 289:22536-53
Michalski, Radoslaw; Michalowski, Bartosz; Sikora, Adam et al. (2014) On the use of fluorescence lifetime imaging and dihydroethidium to detect superoxide in intact animals and ex vivo tissues: a reassessment. Free Radic Biol Med 67:278-84
Kalyanaraman, Balaraman; Dranka, Brian P; Hardy, Micael et al. (2014) HPLC-based monitoring of products formed from hydroethidine-based fluorogenic probes--the ultimate approach for intra- and extracellular superoxide detection. Biochim Biophys Acta 1840:739-44
Sikora, Adam; Zielonka, Jacek; Adamus, Jan et al. (2013) Reaction between peroxynitrite and triphenylphosphonium-substituted arylboronic acid isomers: identification of diagnostic marker products and biological implications. Chem Res Toxicol 26:856-67
Michalski, Radoslaw; Zielonka, Jacek; Hardy, Micael et al. (2013) Hydropropidine: a novel, cell-impermeant fluorogenic probe for detecting extracellular superoxide. Free Radic Biol Med 54:135-47
Zielonka, Jacek; Joseph, Joy; Sikora, Adam et al. (2013) Real-time monitoring of reactive oxygen and nitrogen species in a multiwell plate using the diagnostic marker products of specific probes. Methods Enzymol 526:145-57

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