Regulation of mitochondrial NADP+-dependent isocitrate dehydrogenase activity by glutathionylation.
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.
Kil
In Sup
IS
Department of Biochemistry, College of Natural Sciences, Kyungpook National University, Taegu 702-701, Korea.
Park
Jeen-Woo
JW
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
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