Inactivation of NADP+-dependent isocitrate dehydrogenase by peroxynitrite. Implications for cytotoxicity and alcohol-induced liver injury.
Recently, we demonstrated that the control of cytosolic and mitochondrial redox balance and oxidative damage is one of the primary functions of NADP+-dependent isocitrate dehydrogenase (ICDH) by supplying NADPH for antioxidant systems. We investigated whether the ICDH would be a vulnerable target of peroxynitrite anion (ONOO-) as a purified enzyme, in intact cells, and in liver mitochondria from ethanol-fed rats. Synthetic peroxynitrite and 3-morpholinosydnomine N-ethylcarbamide (SIN-1), a peroxynitrite-generating compound, inactivated ICDH in a dose- and time-dependent manner. The inactivation of ICDH by peroxynitrite or SIN-1 was reversed by dithiothreitol. Loss of enzyme activity was associated with the depletion of the thiol groups in protein. Immunoblotting analysis of peroxynitrite-modified ICDH indicates that S-nitrosylation of cysteine and nitration of tyrosine residues are the predominant modifications. Using electrospray ionization mass spectrometry (ESI-MS) with tryptic digestion of protein, we found that peroxynitrite forms S-nitrosothiol adducts on Cys305 and Cys387 of ICDH. Nitration of Tyr280 was also identified, however, this modification did not significantly affect the activity of ICDH. These results indicate that S-nitrosylation of cysteine residues on ICDH is a mechanism involving the inactivation of ICDH by peroxynitrite. The structural alterations of modified enzyme were indicated by the changes in protease susceptibility and binding of the hydrophobic probe 8-anilino-1-napthalene sulfonic acid. When U937 cells were incubated with 100 microM SIN-1 bolus, a significant decrease in both cytosolic and mitochondrial ICDH activities were observed. Using immunoprecipitation and ESI-MS, we were also able to isolate and positively identify S-nitrosylated and nitrated mitochondrial ICDH from SIN-1-treated U937 cells as well as liver from ethanol-fed rats. Inactivation of ICDH resulted in the pro-oxidant state of cells reflected by an increased level of intracellular reactive oxygen species, a decrease in the ratio of [NADPH]/[NADPH + NADP+], and a decrease in the efficiency of reduced glutathione turnover. The peroxynitrite-mediated damage to ICDH may result in the perturbation of the cellular antioxidant defense mechanisms and subsequently lead to a pro-oxidant condition.
Lee
Jin Hyup
JH
Department of Biochemistry, College of Natural Sciences, Kyungpook National University, Taegu 702-701, Korea.
Yang
Eun Sun
ES
Park
Jeen-Woo
JW
D000818:Animals; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D000431:Ethanol / Q000008:administration & dosage / Q000494:pharmacology; D006801:Humans; D007521:Isocitrate Dehydrogenase / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D008930:Mitochondria, Liver / Q000187:drug effects / Q000201:enzymology*; D024101:Mitochondrial Proteins / Q000378:metabolism; D010545:Peroxides / Q000032:analysis; D030421:Peroxynitrous Acid / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013552:Swine; D014443:Tyrosine / Q000378:metabolism
D024101:Mitochondrial Proteins; D010545:Peroxides; D013438:Sulfhydryl Compounds; D030421:Peroxynitrous Acid; D000431:Ethanol; D014443:Tyrosine; D007521:Isocitrate Dehydrogenase; C039355:isocitrate dehydrogenase (NADP+)