title: Regulation of mitochondrial NADP+-dependent isocitrate dehydrogenase activity by glutathionylation.
abstract: Recently, we demonstrated that the control of mitochondrial redox balance and oxidative damage is one of the primary functions of mitochondrial NADP(+)-dependent isocitrate dehydrogenase (IDPm). Because cysteine residue(s) in IDPm are susceptible to inactivation by a number of thiol-modifying reagents, we hypothesized that IDPm is likely a target for regulation by an oxidative mechanism, specifically glutathionylation. Oxidized glutathione led to enzyme inactivation with simultaneous formation of a mixed disulfide between glutathione and the cysteine residue(s) in IDPm, which was detected by immunoblotting with anti-GSH IgG. The inactivated IDPm was reactivated enzymatically by glutaredoxin2 in the presence of GSH, indicating that the inactivated form of IDPm is a glutathionyl mixed disulfide. Mass spectrometry and site-directed mutagenesis further confirmed that glutathionylation occurs to a Cys(269) of IDPm. The glutathionylated IDPm appeared to be significantly less susceptible than native protein to peptide fragmentation by reactive oxygen species and proteolytic digestion, suggesting that glutathionylation plays a protective role presumably through the structural alterations. HEK293 cells and intact respiring mitochondria treated with oxidants inducing GSH oxidation such as H(2)O(2) or diamide showed a decrease in IDPm activity and the accumulation of glutathionylated enzyme. Using immunoprecipitation with anti-IDPm IgG and immunoblotting with anti-GSH IgG, we were also able to purify and positively identify glutathionylated IDPm from 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated mice, a model for Parkinson's disease. The results of the current study indicate that IDPm activity appears to be modulated through enzymatic glutathionylation and deglutathionylation during oxidative stress.
authors.affiliation: Department of Biochemistry, College of Natural Sciences, Kyungpook National University, Taegu 702-701, Korea.
authors.forename: In Sup
authors.initials: IS
authors.lastname: Kil
authors.forename: Jeen-Woo
authors.initials: JW
authors.lastname: Park
mesh_terms: D015632:1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine / Q000494:pharmacology; D000818:Animals; D001921:Brain / Q000378:metabolism; D002460:Cell Line; D003545:Cysteine / Q000737:chemistry; D053938:DNA Fragmentation; D003958:Diamide / Q000378:metabolism / Q000494:pharmacology; D004220:Disulfides / Q000737:chemistry; D015259:Dopamine Agents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D005978:Glutathione / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D015151:Immunoblotting; D047468:Immunoprecipitation; D007521:Isocitrate Dehydrogenase / Q000378:metabolism*; D013058:Mass Spectrometry; D051379:Mice; D008813:Mice, Inbred ICR; D008928:Mitochondria / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000737:chemistry*; D016877:Oxidants / Q000737:chemistry; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D010100:Oxygen / Q000737:chemistry; D011817:Rabbits; D013438:Sulfhydryl Compounds; D013997:Time Factors
chemical_list: D004220:Disulfides; D015259:Dopamine Agents; D016877:Oxidants; D013438:Sulfhydryl Compounds; D003958:Diamide; D009249:NADP; D015632:1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; D006861:Hydrogen Peroxide; D007521:Isocitrate Dehydrogenase; D005978:Glutathione; D003545:Cysteine; D010100:Oxygen