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Inactivation of NADP+-dependent isocitrate dehydrogenase by peroxynitrite. Implications for cytotoxicity and alcohol-induced liver injury.
J Biol Chem. 2003 Dec 19; 278(51):51360-71.JB

Abstract

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.

Authors+Show Affiliations

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

Pub Type(s)

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

Language

eng

PubMed ID

14551203

Citation

Lee, Jin Hyup, et al. "Inactivation of NADP+-dependent Isocitrate Dehydrogenase By Peroxynitrite. Implications for Cytotoxicity and Alcohol-induced Liver Injury." The Journal of Biological Chemistry, vol. 278, no. 51, 2003, pp. 51360-71.
Lee JH, Yang ES, Park JW. Inactivation of NADP+-dependent isocitrate dehydrogenase by peroxynitrite. Implications for cytotoxicity and alcohol-induced liver injury. J Biol Chem. 2003;278(51):51360-71.
Lee, J. H., Yang, E. S., & Park, J. W. (2003). Inactivation of NADP+-dependent isocitrate dehydrogenase by peroxynitrite. Implications for cytotoxicity and alcohol-induced liver injury. The Journal of Biological Chemistry, 278(51), 51360-71.
Lee JH, Yang ES, Park JW. Inactivation of NADP+-dependent Isocitrate Dehydrogenase By Peroxynitrite. Implications for Cytotoxicity and Alcohol-induced Liver Injury. J Biol Chem. 2003 Dec 19;278(51):51360-71. PubMed PMID: 14551203.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Inactivation of NADP+-dependent isocitrate dehydrogenase by peroxynitrite. Implications for cytotoxicity and alcohol-induced liver injury. AU - Lee,Jin Hyup, AU - Yang,Eun Sun, AU - Park,Jeen-Woo, Y1 - 2003/10/09/ PY - 2003/10/11/pubmed PY - 2004/1/31/medline PY - 2003/10/11/entrez SP - 51360 EP - 71 JF - The Journal of biological chemistry JO - J Biol Chem VL - 278 IS - 51 N2 - 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. SN - 0021-9258 UR - https://www.unboundmedicine.com/medline/citation/14551203/Inactivation_of_NADP+_dependent_isocitrate_dehydrogenase_by_peroxynitrite__Implications_for_cytotoxicity_and_alcohol_induced_liver_injury_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0021-9258(20)75396-2 DB - PRIME DP - Unbound Medicine ER -