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Abstract Title: Protein-centered radicals in lipopolysaccharide-activated macrophages: Does myeloperoxidase have a role? _________ PRIMARY AUTHOR INFORMATION______ Name: Zili Mai Degree: Ph.D. Institution Affiliation: Oklahoma Medical Research Foundation Department: Free Radical Biology and Aging Research Program Address: 825 NE 13th Street, MS-21; Oklahoma City, OK, 73104, United States of America Phone: 405-271-7991 Fax: 405-271-1437 Email: zili-zhai@omrlorg ________ CO-AUTHORS________ Sandra E. Gomez-Mejiba, M.Sc. Michael Kinter, Ph.D. Rebecca A. Fads, B.Sc. Leese J. Deterding, Ph.D. Kenneth B. Tomer, Ph.D. Ronald P. Mason, Ph.D. Dario C. Ramirez, Ph.D. Abstract Myeloperoxidase, the only enzyme that uses H202 to oxidize chloride to HOCl/OCI-, is implicated in a number of inflammatory diseases that are of high public concern. Afthough the roles of a number of reactive oxygen species in redox signaling have been extensively studied, the signaling properties of HOCI produced by myeloperoxidase have not been appreciated. We hypothesized that macrophage activation by lipopolysaccharide (LPS) induces MPO expression that, in turn, produces HOCI, which reacts with specific proteins to form protein-centered radicals. Consequently, if protein-centered radicals are involved in cell activation, the nitrone spin trap 5,5,- dimethy1-1-pyrroline N-oxide (DMPO) would trap them and block macrophage activation. In addition, trapping protein-centered radicals with DMPO will label the proteins with the spin trap, allowing their detection, purification, and identification by immuno-spin trapping with the anti-DMPO antibody [1-3]. We sought to study the location, identity, and consequences of proteins oxidized during activation of macrophages by LPS using immuno-spin trapping with the anti-DMPO antibody [2]. LPS induces activation of macrophages as assessed by evaluating changes in cell morphology, inducible nitric oxide (N0) synthase expression, and nitric oxide production. Concurrently, we observed increased levels of MPO protein, MPO activity, p47phox, chlorotyrosine- and carbonyl-proteins, suggesting that HOCI or derived chioramines may be formed during cell activation. When dialyzed homogenates from LPS-treated macrophages were incubated with micmmolar concentrations of H202 and 140 mM CI- in the presence of DMPO, protein-centered radicals were produced, whereas no radical adducts were observed in the absence of Cl- or in the presence of MPO inhibitors. Morphological changes and production of chlorotyrosine-lcarbonyl-proteins and nNO were inhibited by DMPO, suggesting that protein-centered radicals might be important for cell activation. Confocal imaging showed nitrone adducts that were localized dose to MPO in the macrophages activated by LPS. In addition, Western blot analysis showed at least 14 distinctive positive nitrone adduct bands, and the proteins contained in them were identified by LC-MS/MS [2]. To our knowledge, this is the first study that demonstrates MPO-induced protein-centered radicals and their location, identity, and consequences in macrophage activation by LPS. Supported by ROOES015415 (NIEHS). References 1 Ramirez, D. C. and Mason, R. P. (2005) Immuno-spin trapping: detection of protein-centered radicals. In: Cum Protoc. Toxicol. (Costa, L. G., Maines, M. D., Reed, D. J., Sassa, S. and Sipes, I. G., eds.). pp. 17.17.11-17.17.16, John Wiley & Sons, Hoboken, New Jersey, USA 2 Gomez-Mejiba, S. E., Zhai, Z., Akram, H., Deterding, L. J., Hensley, K., Smith, N., Towner, R. A., Tamer, K. B., Mason, R. P. and Ramirez, D. C. (2009) Immuno-spin trapping of protein and DNA radicals: "Tagging" free radicals to locate and understand the redox process. Free Radic. Biol. Med. In Press. 3 Ramirez, D. C., Gomez-Mejiba, S. E. and Mason, R. P. (2007) Immuno-spin trapping analyses ofDNA radicals. Nat. Protoc. 2, 512-522

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