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Regulation of mitochondrial NADP+-dependent isocitrate dehydrogenase activity by glutathionylation.
J Biol Chem. 2005 Mar 18; 280(11):10846-54.JB

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+Show Affiliations

Department of Biochemistry, College of Natural Sciences, Kyungpook National University, Taegu 702-701, Korea.No affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

15653693

Citation

Kil, In Sup, and Jeen-Woo Park. "Regulation of Mitochondrial NADP+-dependent Isocitrate Dehydrogenase Activity By Glutathionylation." The Journal of Biological Chemistry, vol. 280, no. 11, 2005, pp. 10846-54.
Kil IS, Park JW. Regulation of mitochondrial NADP+-dependent isocitrate dehydrogenase activity by glutathionylation. J Biol Chem. 2005;280(11):10846-54.
Kil, I. S., & Park, J. W. (2005). Regulation of mitochondrial NADP+-dependent isocitrate dehydrogenase activity by glutathionylation. The Journal of Biological Chemistry, 280(11), 10846-54.
Kil IS, Park JW. Regulation of Mitochondrial NADP+-dependent Isocitrate Dehydrogenase Activity By Glutathionylation. J Biol Chem. 2005 Mar 18;280(11):10846-54. PubMed PMID: 15653693.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Regulation of mitochondrial NADP+-dependent isocitrate dehydrogenase activity by glutathionylation. AU - Kil,In Sup, AU - Park,Jeen-Woo, Y1 - 2005/01/14/ PY - 2005/1/18/pubmed PY - 2005/4/26/medline PY - 2005/1/18/entrez SP - 10846 EP - 54 JF - The Journal of biological chemistry JO - J Biol Chem VL - 280 IS - 11 N2 - 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. SN - 0021-9258 UR - https://www.unboundmedicine.com/medline/citation/15653693/Regulation_of_mitochondrial_NADP+_dependent_isocitrate_dehydrogenase_activity_by_glutathionylation_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0021-9258(19)30493-4 DB - PRIME DP - Unbound Medicine ER -