GAPDH-centered radicals in LPS-triggered activation of macrophages

ZILI ZHAI; SANDRA E. GOMEZ-MEJIBA; GANGA MOORTHY; REBECCA FARIS; MICHAEL KINTER; DARIO C. RAMIREZ

New York, NY

Conferencia; The 4th International Conference on Oxidative/nitrosative stress and diseases; 2009

The New York Academy of Sciences

GAPDH-CENTERED RADICALS IN LPS-TRIGGERED ACTIVATION OF MACROPHAGES   Zili Zhai, Ph.D., Sandra E. Gomez-Mejiba, M.Sc., Ganga Moorthy, B.Sc., Rebecca A. Faris, B.F.A., Michael T. Kinter, Ph.D., and Dario C. Ramirez, Ph.D. M.Sc., Free Radical Biology and Aging Research Program, Oklahoma Medical Research Foundation. Oklahoma City, OK 73104   The nitrone spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) traps free radicals, including protein-centered radicals to form protein-DMPO nitrone adducts that we can study using immuno-spin trapping. Interestingly, lipopolysaccharide (LPS)-induced protein oxidation, activation and death of RAW 264.7 macrophages were prevented by DMPO. We sought to test whether protein-centered radicals are produced during LPS-triggered activation of macrophages. We observed that DMPO traps a number of protein-centered radicals; most of them were localized in the perinuclear region of the macrophage. This is a typical localization of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in stressed cells. Immunoprecipitation of these nitrone adducts with an anti-DMPO immuno-specific molecular catcher and subsequent SDS-PAGE showed, among other bands, a major band of proteins at ~ 38 kDa. The tandem-MS and Western blot analysis identified GAPDH in this band. Western blot with the polyclonal anti-DMPO showed a GAPDH-DMPO nitrone adduct. Incubation of the purified rabbit muscle GAPDH with HOCl and DMPO formed GAPDH-DMPO nitrone adducts, whereas H2O2, acetaldehyde/xanthine oxidase, or SIN-1 (a source of peroxinitrite) did not, suggesting that HOCl might be the oxidant that forms GAPDH-centered radicals in LPS-activated macrophages. In addition it is known HOCl and chloramines inactivate GAPDH. Interestingly, GAPDH and MPO co-localize in the perinuclear region, where most of the nitrone adducts were trapped with DMPO during the activation of macrophages. Overall, our results indicate a possible role of MPO-driven, HOCl-mediated oxidative modifications of GAPDH in the activation and death signaling triggered by LPS in macrophages. Supported by NIEHS 5R00ES015415-03.