Publications by authors named "In Sup Kil"

34 Publications

Extracellular Vesicles Derived from Senescent Fibroblasts Attenuate the Dermal Effect on Keratinocyte Differentiation.

Int J Mol Sci 2020 Feb 4;21(3). Epub 2020 Feb 4.

Basic Research & Innovation Division, R&D Unit, AmorePacific Corporation, Yongin 17074, Korea.

The skin is a multilayered and primary defensive organ. Intimate intercellular communication in the skin is necessary to ensure effective surveillance. Extracellular vesicles (EVs) are being explored for their involvement in intercellular skin communication. The aim of this study was to evaluate how human dermal fibroblasts (HDFs) accelerate EV production during senescence and the effects of senescence-associated EVs on epidermal homeostasis. Replicative senescent HDFs were assessed with senescence-associated β-galactosidase staining and the expression of senescence-related markers. Isolated EVs were characterized by dynamic light scattering and EV marker expression. EVs secreted from untreated young or senescent HDFs, or from those treated with a nSMase inhibitor, antioxidant, and lysosomal activity regulators, were determined by sandwich ELISA for CD81. Human epidermal keratinocytes were treated with young- and senescent HDF-derived EVs. Compared to young HDFs, senescent HDFs produced relatively high levels of EVs due to the increased nSMase activity, oxidative stress, and altered lysosomal activity. The nSMase inhibitor, antioxidant, and agents that recovered lysosomal activity reduced EV secretion in senescent HDFs. Relative to young HDF-derived EVs, senescent HDF-derived EVs were less supportive in keratinocyte differentiation and barrier function but increased proinflammatory cytokine IL-6 levels. Our study suggests that dermis-derived EVs may regulate epidermal homeostasis by reflecting cellular status, which provides insight as to how the dermis communicates with the epidermis and influences skin senescence.
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http://dx.doi.org/10.3390/ijms21031022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7037765PMC
February 2020

Oxalomalate reduces tumor progression in melanoma via ROS-dependent proapoptotic and antiangiogenic effects.

Biochimie 2019 Mar 9;158:165-171. Epub 2019 Jan 9.

School of Life Sciences and Biotechnology, BK21 Plus KNU Creative BioResearch Group, College of Natural Sciences, Kyungpook National University, Taegu, South Korea. Electronic address:

The potent cytotoxicity of reactive oxygen species (ROS) can cause various diseases, however, it may also serve as a powerful chemotherapeutic strategy capable of killing cancer cells. Oxalomalate (OMA, α-hydroxy-β-oxalosuccinic acid), a tricarboxylic acid intermediate, is a well-known competitive inhibitor of two classes of NADP-dependent isocitrate dehydrogenase (IDH) isoenzymes, which serve as the major antioxidants and redox regulators in the mitochondria and cytosol. In this study, we investigated the therapeutic effects of OMA in melanoma and elucidated the associated underlying mechanisms of action using in vitro and in vivo models. OMA targeting IDH enzymes suppressed melanoma growth through activation of apoptosis and inhibition of angiogenesis. Mechanistically, our findings showed that OMA activated p53-mediated apoptosis through ROS-dependent ATM-Chk2 signaling and reduced the expression of vascular endothelial growth factor through ROS-dependent E2F1-mediated hypoxia inducible factor-1α degradation. In particular, OMA-induced suppression of IDH activity resulted in induction of ROS stress response, ultimately leading to apoptotic cell death and antiangiogenic effects in melanoma cells. Thus, OMA might be a potential candidate drug for melanoma skin cancer therapy.
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http://dx.doi.org/10.1016/j.biochi.2019.01.004DOI Listing
March 2019

Peroxiredoxin5 Controls Vertebrate Ciliogenesis by Modulating Mitochondrial Reactive Oxygen Species.

Antioxid Redox Signal 2019 05 30;30(14):1731-1745. Epub 2018 Oct 30.

1 KNU-Center for Nonlinear Dynamics, CMRI, School of Life Sciences, BK21 Plus KNU Creative BioResearch Group, College of Natural Sciences, Kyungpook National University , Daegu, South Korea .

Aims: Peroxiredoxin5 (Prdx5), a thioredoxin peroxidase, is an antioxidant enzyme that is widely studied for its antioxidant properties and protective roles in neurological and cardiovascular disorders. This study is aimed at investigating the functional significance of Prdx5 in mitochondria and at analyzing its roles in ciliogenesis during the process of vertebrate development.

Results: We found that several Prdx genes were strongly expressed in multiciliated cells in developing Xenopus embryos, and their peroxidatic functions were crucial for normal cilia development. Depletion of Prdx5 increased levels of cellular reactive oxygen species (ROS), consequently leading to mitochondrial dysfunction and abnormal cilia formation. Proteomic and transcriptomic approaches revealed that excessive ROS accumulation on Prdx5 depletion subsequently reduced the expression level of pyruvate kinase (PK), a key metabolic enzyme in energy production. We further confirmed that the promotor activity of PK was significantly reduced on Prdx5 depletion and that the reduction in PK expression and its promoter activity led to ciliary defects observed in Prdx5-depleted cells.

Innovation: Our data revealed the novel relationship between ROS and Prdx5 and the consequent effects of this interaction on vertebrate ciliogenesis. The normal process of ciliogenesis is interrupted by the Prdx5 depletion, resulting in excessive ROS levels and suggesting cilia as vulnerable targets of ROS.

Conclusion: Prdx5 plays protective roles in mitochondria and is critical for normal cilia development by regulating the levels of ROS. The loss of Prdx5 is associated with excessive production of ROS, resulting in mitochondrial dysfunction and aberrant ciliogenesis.
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http://dx.doi.org/10.1089/ars.2018.7507DOI Listing
May 2019

Pyruvate Protects against Cellular Senescence through the Control of Mitochondrial and Lysosomal Function in Dermal Fibroblasts.

J Invest Dermatol 2018 12 28;138(12):2522-2530. Epub 2018 Jun 28.

Basic Research & Innovation Division, Amorepacific Corporation R&D Center, Yongin-si, Gyeonggi-do, Republic of Korea. Electronic address:

Mitochondrial dysfunction can drive cellular senescence, which is accompanied by changes in metabolism and increases in senescence-associated secretory phenotypes. Although pyruvate, a key metabolite for numerous aspects of metabolism, has been used as general supplement in synthetic media, the physiological function of pyruvate underlying its protective role against cellular senescence under normal conditions has remained unknown. Here, we show that extracellular pyruvate prevents senescence in normal human dermal fibroblasts through increasing the generation of oxidized nicotinamide adenine dinucleotide (NAD) during the conversion to lactate. Acetylated peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α), vacuolar-type H-ATPaseV0A1 (v-ATPaseV0A1), NF-κB p65 subunit (RelA), and histone H3 accumulate under pyruvate deprivation conditions, resulting in the onset of senescence in normal human dermal fibroblasts through the accumulation of abnormal mitochondria generated by lysosomal inactivation-induced mitophagy defects, and through an increase in senescence-associated secretory phenotypes. Furthermore, pyruvate showed a protective effect against aging phenotypes in skin equivalents, which consist of a dermis and epidermis that act similarly to in vivo skin tissues. Our findings reveal a connection between pyruvate and mitochondrial dysfunction in the progression of senescence that is, to our knowledge, previously unreported. These results suggest that the pyruvate deprivation-induced senescence model can be used to study the connection between metabolism and senescence under normal conditions.
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http://dx.doi.org/10.1016/j.jid.2018.05.033DOI Listing
December 2018

Mitochondrial HO signaling is controlled by the concerted action of peroxiredoxin III and sulfiredoxin: Linking mitochondrial function to circadian rhythm.

Free Radic Biol Med 2016 11 27;100:73-80. Epub 2016 Oct 27.

Yonsei Biomedical Research Institute, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752, Republic of Korea.

Mitochondria produce hydrogen peroxide (HO) during energy metabolism in most mammalian cells as well as during the oxidation of cholesterol associated with the synthesis of steroid hormones in steroidogenic cells. Some of the HO produced in mitochondria is released into the cytosol, where it serves as a key regulator of various signaling pathways. Given that mitochondria are equipped with several HO-eliminating enzymes, however, it had not been clear how mitochondrial HO can escape destruction by these enzymes for such release. Peroxiredoxin III (PrxIII) is the most abundant and efficient HO-eliminating enzyme in mitochondria of most cell types. We found that PrxIII undergoes reversible inactivation through hyperoxidation of its catalytic cysteine residue to cysteine sulfinic acid, and that release of mitochondrial HO likely occurs as a result of such PrxIII inactivation. The hyperoxidized form of PrxIII (PrxIII-SOH) is reduced and reactivated by sulfiredoxin (Srx). We also found that the amounts of PrxIII-SOH and Srx undergo antiphasic circadian oscillation in mitochondria of the adrenal gland, heart, and brown adipose tissue of mice maintained under normal conditions. Cytosolic Srx was found to be imported into mitochondria via a mechanism that requires formation of a disulfide-linked complex with heat shock protein 90, which is likely promoted by HO released from mitochondria. The imported Srx was found to be degraded by Lon protease in a manner dependent on PrxIII hyperoxidation state. The coordinated import and degradation of Srx underlie Srx oscillation and consequent PrxIII-SOH oscillation in mitochondria. The rhythmic change in the amount of PrxIII-SOH suggests that mitochondrial release of HO is also likely a circadian event that conveys temporal information on steroidogenesis in the adrenal gland and on energy metabolism in heart and brown adipose tissue to cytosolic signaling pathways.
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http://dx.doi.org/10.1016/j.freeradbiomed.2016.10.011DOI Listing
November 2016

Multiple Functions and Regulation of Mammalian Peroxiredoxins.

Annu Rev Biochem 2017 06 2;86:749-775. Epub 2017 Feb 2.

Yonsei Biomedical Research Institute, Yonsei University College of Medicine, Seoul 120-752, Korea; email:

Peroxiredoxins (Prxs) constitute a major family of peroxidases, with mammalian cells expressing six Prx isoforms (PrxI to PrxVI). Cells produce hydrogen peroxide (HO) at various intracellular locations where it can serve as a signaling molecule. Given that Prxs are abundant and possess a structure that renders the cysteine (Cys) residue at the active site highly sensitive to oxidation by HO, the signaling function of this oxidant requires extensive and highly localized regulation. Recent findings on the reversible regulation of PrxI through phosphorylation at the centrosome and on the hyperoxidation of the Cys at the active site of PrxIII in mitochondria are described in this review as examples of such local regulation of HO signaling. Moreover, their high affinity for and sensitivity to oxidation by HO confer on Prxs the ability to serve as sensors and transducers of HO signaling through transfer of their oxidation state to bound effector proteins.
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http://dx.doi.org/10.1146/annurev-biochem-060815-014431DOI Listing
June 2017

Mitochondrial HO signaling is controlled by the concerted action of peroxiredoxin III and sulfiredoxin: Linking mitochondrial function to circadian rhythm.

Free Radic Biol Med 2016 10 4;99:120-127. Epub 2016 Aug 4.

Yonsei Biomedical Research Institute, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752, Republic of Korea.

Mitochondria produce hydrogen peroxide (HO) during energy metabolism in most mammalian cells as well as during the oxidation of cholesterol associated with the synthesis of steroid hormones in steroidogenic cells. Some of the HO produced in mitochondria is released into the cytosol, where it serves as a key regulator of various signaling pathways. Given that mitochondria are equipped with several HO-eliminating enzymes, however, it had not been clear how mitochondrial HO can escape destruction by these enzymes for such release. Peroxiredoxin III (PrxIII) is the most abundant and efficient HO-eliminating enzyme in mitochondria of most cell types. We found that PrxIII undergoes reversible inactivation through hyperoxidation of its catalytic cysteine residue to cysteine sulfinic acid, and that release of mitochondrial HO likely occurs as a result of such PrxIII inactivation. The hyperoxidized form of PrxIII (PrxIII-SOH) is reduced and reactivated by sulfiredoxin (Srx). We also found that the amounts of PrxIII-SOH and Srx undergo antiphasic circadian oscillation in mitochondria of the adrenal gland, heart, and brown adipose tissue of mice maintained under normal conditions. Cytosolic Srx was found to be imported into mitochondria via a mechanism that requires formation of a disulfide-linked complex with heat shock protein 90, which is likely promoted by HO released from mitochondria. The imported Srx was found to be degraded by Lon protease in a manner dependent on PrxIII hyperoxidation state. The coordinated import and degradation of Srx underlie Srx oscillation and consequent PrxIII-SOH oscillation in mitochondria. The rhythmic change in the amount of PrxIII-SOH suggests that mitochondrial release of HO is also likely a circadian event that conveys temporal information on steroidogenesis in the adrenal gland and on energy metabolism in heart and brown adipose tissue to cytosolic signaling pathways.
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http://dx.doi.org/10.1016/j.freeradbiomed.2016.07.029DOI Listing
October 2016

Circadian Oscillation of Sulfiredoxin in the Mitochondria.

Mol Cell 2015 Aug 30;59(4):651-63. Epub 2015 Jul 30.

Yonsei Biomedical Research Institute, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752, Korea. Electronic address:

Hydrogen peroxide (H2O2) released from mitochondria regulates various cell signaling pathways. Given that H2O2-eliminating enzymes such as peroxiredoxin III (PrxIII) are abundant in mitochondria, however, it has remained unknown how such release can occur. Active PrxIII-SH undergoes reversible inactivation via hyperoxidation to PrxIII-SO2, which is then reduced by sulfiredoxin. We now show that the amounts of PrxIII-SO2 and sulfiredoxin undergo antiphasic circadian oscillation in the mitochondria of specific tissues of mice maintained under normal conditions. Cytosolic sulfiredoxin was found to be imported into the mitochondria via a mechanism that requires formation of a disulfide-linked complex with heat shock protein 90, which is promoted by H2O2 released from mitochondria. The imported sulfiredoxin is degraded by Lon in a manner dependent on PrxIII hyperoxidation state. The coordinated import and degradation of sulfiredoxin provide the basis for sulfiredoxin oscillation and consequent PrxIII-SO2 oscillation in mitochondria and likely result in an oscillatory H2O2 release.
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http://dx.doi.org/10.1016/j.molcel.2015.06.031DOI Listing
August 2015

S-Nitrosylation of p47(phox) enhances phosphorylation by casein kinase 2.

Redox Rep 2015 Sep 27;20(5):228-33. Epub 2015 May 27.

Objectives: Leukocyte NADPH oxidase, which is active in neutrophils, is a membrane-bound enzyme that catalyzes the reduction of oxygen to O2(-) by using NADPH as an electron donor. Previously, we reported that casein kinase 2 (CK2), a ubiquitous and highly conserved Ser/Thr kinase, is responsible for p47(phox) phosphorylation and that phosphorylation of p47(phox) by CK2 regulates the deactivation of NADPH oxidase.

Methods: Here, we report that the residue Cys(196) of p47(phox) is a target of S-nitrosylation by S-nitrosothiol and peroxynitrite and that this modification enhanced phosphorylation of p47(phox) by CK2.

Results: S-Nitrosylated p47(phox) enhanced CK2 b subunit binding, presumably due to alterations in protein conformation.

Discussion: Taken together, we propose that S-nitrosylation of p47(phox) regulates the deactivation of NADPH oxidase via enhancement of p47(phox) phosphorylation by CK2.
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http://dx.doi.org/10.1179/1351000215Y.0000000014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6837347PMC
September 2015

Study of the signaling function of sulfiredoxin and peroxiredoxin III in isolated adrenal gland: unsuitability of clonal and primary adrenocortical cells.

Methods Enzymol 2013 ;527:169-81

Yonsei Biomedical Research Institute, Yonsei University College of Medicine, Seodaemun-gu, Seoul, South Korea.

Members of the peroxiredoxin (Prx) family of antioxidant enzymes are inactivated via hyperoxidation of the active site cysteine by the substrate H2O2 and are reactivated via an ATP-consuming process catalyzed by sulfiredoxin (Srx). PrxIII is reversibly inactivated by H2O2 produced by cytochrome P450 11B1 (CYP11B1) in mitochondria during corticosterone synthesis in the adrenal gland of mice injected with adrenocorticotropic hormone (ACTH). Inactivation of PrxIII triggers a sequence of events including accumulation of H2O2, activation of p38 mitogen-activated kinase (MAPK), inhibition of cholesterol transfer, and suppression of corticosterone synthesis. Srx expression is significantly induced by ACTH injection. The coupling of CYP11B1 activity to PrxIII inactivation and Srx induction provides a feedback regulatory mechanism for steroidogenesis that functions independently of the hypothalamic-pituitary-adrenal axis. Furthermore, the PrxIII-Srx regulatory pathway is critical for the circadian rhythm of corticosterone production. Although adrenocortical tumor cell lines such as Y-1 and H295R have been used extensively for studying the mechanism of steroidogenesis, those clonal cells were found to be unsuitable as an in vitro model for redox signaling because the amount of Srx in the cell lines is much higher than that in mouse adrenal gland and not affected by ACTH stimulation. Furthermore, the levels of PrxIII in the clonal cells are greatly reduced compared to that in the adrenal gland, and ACTH does not induce PrxIII hyperoxidation in the clonal cells. Primary adrenocortical cells isolated from the mouse adrenal gland were also found to be an invalid model because Srx levels are increased, along with decreased levels of hyperoxidized PrxIII, soon after isolation of these cells. Organ culture system is, however, appropriate for studying the PrxIII-Srx regulatory function as the levels of hyperoxidized PrxIII and Srx in the adrenal glands maintained overnight in culture medium are not changed.
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http://dx.doi.org/10.1016/B978-0-12-405882-8.00009-XDOI Listing
January 2014

S-glutathionylation regulates GTP-binding of Rac2.

Biochem Biophys Res Commun 2012 Sep 7;425(4):892-6. Epub 2012 Aug 7.

School of Life Sciences and Biotechnology, College of Natural Sciences, Kyungpook National University, Taegu 702701, Republic of Korea.

Phagocyte NADPH oxidase catalyzes the reduction of molecular oxygen to superoxide and is essential for defense against microbes. Rac2 is a low molecular weight GTP-binding protein that has been implicated in the regulation of phagocyte NADPH oxidase. Here we report that Cys(157) of Rac2 is a target of S-glutathionylation and that this modification is reversed by dithiothreitol as well as enzymatically by thioltransferase in the presence of GSH. S-glutathionylated Rac2 enhanced the binding of GTP, presumably due to structural alterations. These results elucidate the redox regulation of cysteine in Rac2 and a possible mechanism for regulating NADPH oxidase activation.
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http://dx.doi.org/10.1016/j.bbrc.2012.07.169DOI Listing
September 2012

Feedback control of adrenal steroidogenesis via H2O2-dependent, reversible inactivation of peroxiredoxin III in mitochondria.

Mol Cell 2012 Jun;46(5):584-94

Division of Life and Pharmaceutical Sciences, Ewha Womans University, Seoul, Korea.

Certain members of the peroxiredoxin (Prx) family undergo inactivation through hyperoxidation of the catalytic cysteine to sulfinic acid during catalysis and are reactivated by sulfiredoxin; however, the physiological significance of this reversible regulatory process is unclear. We now show that PrxIII in mouse adrenal cortex is inactivated by H(2)O(2) produced by cytochrome P450 enzymes during corticosterone production stimulated by adrenocorticotropic hormone. Inactivation of PrxIII triggers a sequence of events including accumulation of H(2)O(2), activation of p38 mitogen-activated protein kinase, suppression of steroidogenic acute regulatory protein synthesis, and inhibition of steroidogenesis. Interestingly, levels of inactivated PrxIII, activated p38, and sulfiredoxin display circadian oscillations. Steroidogenic tissue-specific ablation of sulfiredoxin in mice resulted in the persistent accumulation of inactive PrxIII and suppression of the adrenal circadian rhythm of corticosterone production. The coupling of CYP11B1 activity to PrxIII inactivation provides a feedback regulatory mechanism for steroidogenesis that functions independently of the hypothalamic-pituitary-adrenal axis.
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http://dx.doi.org/10.1016/j.molcel.2012.05.030DOI Listing
June 2012

Peroxiredoxin III and sulfiredoxin together protect mice from pyrazole-induced oxidative liver injury.

Antioxid Redox Signal 2012 Nov 31;17(10):1351-61. Epub 2012 May 31.

Division of Life and Pharmaceutical Sciences, Ewha Womans University, Seoul, Korea.

Aims: To define the mechanisms underlying pyrazole-induced oxidative stress and the protective role of peroxiredoxins (Prxs) and sulfiredoxin (Srx) against such stress.

Results: Pyrazole increased Srx expression in the liver of mice in a nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent manner and induced Srx translocation from the cytosol to the endoplasmic reticulum (ER) and mitochondria. Pyrazole also induced the expression of CYP2E1, a primary reactive oxygen species (ROS) source for ethanol-induced liver injury, in ER and mitochondria. However, increased CYP2E1 levels only partially accounted for the pyrazole-mediated induction of Srx, prompting the investigation of CYP2E1-independent ROS generation downstream of pyrazole. Indeed, pyrazole increased ER stress, which is known to elevate mitochondrial ROS. In addition, pyrazole up-regulated CYP2E1 to a greater extent in mitochondria than in ER. Accordingly, among Prxs I to IV, PrxIII, which is localized to mitochondria, was preferentially hyperoxidized in the liver of pyrazole-treated mice. Pyrazole-induced oxidative damage to the liver was greater in PrxIII(-/-) mice than in wild-type mice. Such damage was also increased in Srx(-/-) mice treated with pyrazole, underscoring the role of Srx as the guardian of PrxIII.

Innovation: The roles of Prxs, Srx, and ER stress have not been previously studied in relation to pyrazole toxicity.

Conclusion: The concerted action of PrxIII and Srx is important for protection against pyrazole-induced oxidative stress arising from the convergent induction of CYP2E1-derived and ER stress-derived ROS in mitochondria.
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http://dx.doi.org/10.1089/ars.2011.4334DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3437045PMC
November 2012

Peroxiredoxin functions as a peroxidase and a regulator and sensor of local peroxides.

J Biol Chem 2012 Feb 6;287(7):4403-10. Epub 2011 Dec 6.

Division of Life and Pharmaceutical Sciences, Ewha Womans University,Seoul 120-750, Korea.

Peroxiredoxins (Prxs) contain an active site cysteine that is sensitive to oxidation by H(2)O(2). Mammalian cells express six Prx isoforms that are localized to various cellular compartments. The oxidized active site cysteine of Prx can be reduced by a cellular thiol, thus enabling Prx to function as a locally constrained peroxidase. Regulation of Prx via phosphorylation in response to extracellular signals allows the local accumulation of H(2)O(2) and thereby enables its messenger function. The fact that the oxidation state of the active site cysteine of Prx can be transferred to other proteins that are less intrinsically susceptible to H(2)O(2) also allows Prx to function as an H(2)O(2) sensor.
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http://dx.doi.org/10.1074/jbc.R111.283432DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3281607PMC
February 2012

Attenuated mitochondrial NADP+-dependent isocitrate dehydrogenase activity enhances EGCG-induced apoptosis.

Biochimie 2011 Oct 2;93(10):1808-15. Epub 2011 Jul 2.

School of Life Sciences and Biotechnology, College of Natural Sciences, Kyungpook National University, Taegu 702-701, South Korea.

(-)-Epigallocatechin-3-gallate (EGCG), a well-known chemopreventive factor, induces cancer cells undergoing apoptosis. Over the last several years, we have shown that the mitochondrial NADP(+)-dependent isocitrate dehydrogenase (IDPm) functions as an antioxidant and anti-apoptotic protein by supplying NADPH to antioxidant systems. Here, we show that EGCG induced the inactivation of IDPm as a purified enzyme and in cultured cancer cells in a dose- and time-dependent manner. Loss of enzyme activity was associated with the depletion of the thiol groups in protein. In addition, transfection of HeLa cells with an IDPm small interfering RNA (siRNA) markedly attenuated the activity of IDPm and substantially enhanced EGCG-induced apoptosis as indicated by the morphological evidence of apoptosis, DNA fragmentation, and the modulation of mitochondrial function and apoptotic marker proteins. Taken together, our results suggest that the suppression of IDPm activity resulted in the disruption of cellular redox balance and subsequently exacerbates EGCG-induced apoptotic cell death in HeLa cells. These results might have implications for developing an effective combination modality in cancer treatment.
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http://dx.doi.org/10.1016/j.biochi.2011.06.025DOI Listing
October 2011

Concerted action of sulfiredoxin and peroxiredoxin I protects against alcohol-induced oxidative injury in mouse liver.

Hepatology 2011 Mar 11;53(3):945-53. Epub 2011 Feb 11.

Department of Life Science, Division of Life and Pharmaceutical Sciences, Ewha Womans University, Seoul, Korea.

Unlabelled: Peroxiredoxins (Prxs) are peroxidases that catalyze the reduction of reactive oxygen species (ROS). The active site cysteine residue of members of the 2-Cys Prx subgroup (Prx I to IV) of Prxs is hyperoxidized to cysteine sulfinic acid (Cys-SO(2) ) during catalysis with concomitant loss of peroxidase activity. Reactivation of the hyperoxidized Prx is catalyzed by sulfiredoxin (Srx). Ethanol consumption induces the accumulation of cytochrome P450 2E1 (CYP2E1), a major contributor to ethanol-induced ROS production in the liver. We now show that chronic ethanol feeding markedly increased the expression of Srx in the liver of mice in a largely Nrf2-dependent manner. Among Prx I to IV, only Prx I was found to be hyperoxidized in the liver of ethanol-fed wildtype mice, and the level of Prx I-SO(2) increased to ≈30% to 50% of total Prx I in the liver of ethanol-fed Srx(-/-) mice. This result suggests that Prx I is the most active 2-Cys Prx in elimination of ROS from the liver of ethanol-fed mice and that, despite the up-regulation of Srx expression by ethanol, the capacity of Srx is not sufficient to counteract the hyperoxidation of Prx I that occurs during ROS reduction. A protease protection assay revealed that a large fraction of Prx I is located together with CYP2E1 at the cytosolic side of the endoplasmic reticulum membrane. The selective role of Prx I in ROS removal is thus likely attributable to the proximity of Prx I and CYP2E1.

Conclusion: The pivotal functions of Srx and Prx I in protection of the liver in ethanol-fed mice was evident from the severe oxidative damage observed in mice lacking either Srx or Prx I.
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http://dx.doi.org/10.1002/hep.24104DOI Listing
March 2011

Silencing of mitochondrial NADP+-dependent isocitrate dehydrogenase gene enhances selenite-induced apoptosis.

Free Radic Res 2010 Mar;44(3):332-9

School of Life Sciences and Biotechnology, College of Natural Sciences, Kyungpook National University, Taegu 702-701, South Korea.

Selenium has been shown to play a chemopreventive role in human cancer, presumably by inducing tumour cell apoptosis. Selenite is thought to induce oxidative stress by the generation of the superoxide anion and catalysing the oxidation of thiol groups. It has previously been reported that control of the mitochondrial redox balance is a primary function of mitochondrial NADP(+)-dependent isocitrate dehydrogenase (IDPm) by supplying NADPH for antioxidant systems. When investigating whether IDPm would be a vulnerable target of selenite, the loss of enzyme activity was observed. Transfection of HeLa cells with an IDPm small interfering RNA (siRNA) markedly decreased activity of IDPm and enhanced cells' susceptibility of selenite-induced apoptosis, as indicated by morphological evidence of apoptosis, DNA fragmentation and the modulation of mitochondrial function and apoptotic marker proteins. These results suggest that IDPm siRNA sensitizes HeLa cells to selenite-induced apoptotic cell death, presumably through the perturbation of the cellular redox status.
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http://dx.doi.org/10.3109/10715760903494184DOI Listing
March 2010

Cytosolic NADP(+)-dependent isocitrate dehydrogenase regulates cadmium-induced apoptosis.

Biochem Pharmacol 2010 Apr 26;79(7):1072-80. Epub 2009 Nov 26.

School of Life Sciences and Biotechnology, College of Natural Sciences, Kyungpook National University, Taegu 702-701, Republic of Korea.

Cadmium ions have a high affinity for thiol groups. Therefore, they may disturb many cellular functions. We recently reported that cytosolic NADP(+)-dependent isocitrate dehydrogenase (IDPc) functions as an antioxidant enzyme to supply NADPH, a major source of reducing equivalents to the cytosol. Cadmium decreased the activity of IDPc both as a purified enzyme and in cultured cells. In the present study, we demonstrate that the knockdown of IDPc expression in HEK293 cells greatly enhances apoptosis induced by cadmium. Transfection of HEK293 cells with an IDPc small interfering RNA significantly decreased the activity of IDPc and enhanced cellular susceptibility to cadmium-induced apoptosis as indicated by the morphological evidence of apoptosis, DNA fragmentation and condensation, cellular redox status, mitochondria redox status and function, and the modulation of apoptotic marker proteins. Taken together, our results suggest that suppressing the expression of IDPc enhances cadmium-induced apoptosis of HEK293 cells by increasing disruption of the cellular redox status.
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http://dx.doi.org/10.1016/j.bcp.2009.11.014DOI Listing
April 2010

Glutathionylation regulates cytosolic NADP+-dependent isocitrate dehydrogenase activity.

Free Radic Res 2009 Apr 17;43(4):409-16. Epub 2009 Mar 17.

School of Life Sciences and Biotechnology, College of Natural Sciences, Kyungpook National University, Taegu 702-701, Korea.

Cytosolic NADP+-dependent isocitrate dehydrogenase (IDPc) is susceptible to inactivation by numerous thiol-modifying reagents. This study now reports that Cys269 of IDPc is a target for S-glutathionylation and that this modification is reversed by dithiothreitol as well as enzymatically by cytosolic glutaredoxin in the presence of GSH. Glutathionylated IDPc was significantly less susceptible than native protein to peptide fragmentation by reactive oxygen species and proteolytic digestion. Glutathionylation may play a protective role in the degradation of protein through the structural alterations of IDPc. HEK293 cells treated with diamide displayed decreased IDPc activity and accumulated glutathionylated enzyme. Using immunoprecipitation with an anti-IDPc IgG and immunoblotting with an anti-GSH IgG, we purified and positively identified glutathionylated IDPc from the kidneys of mice subjected to ischemia/reperfusion injury and from the livers of ethanol-administered rats. These results suggest that IDPc activity is modulated through enzymatic glutathionylation and deglutathionylation during oxidative stress.
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http://dx.doi.org/10.1080/10715760902801525DOI Listing
April 2009

Silencing of cytosolic NADP(+)-dependent isocitrate dehydrogenase by small interfering RNA enhances the sensitivity of HeLa cells toward staurosporine.

Free Radic Res 2009 Feb;43(2):165-73

College of Natural Sciences, Kyungpook National University, Taegu, Korea.

Staurosporine induces the production of reactive oxygen species, which play an important causative role in apoptotic cell death. Recently, it was demonstrated that the control of cellular redox balance and the defense against oxidative damage is one of the primary functions of cytosolic NADP(+)-dependent isocitrate dehydrogenase (IDPc) by supplying NADPH for antioxidant systems. The present report shows that silencing of IDPc expression in HeLa cells greatly enhances apoptosis induced by staurosporine. Transfection of HeLa cells with an IDPc small interfering RNA (siRNA) markedly decreased activity of IDPc, enhancing the susceptibility of staurosporine-induced apoptosis reflected by DNA fragmentation, cellular redox status and the modulation of apoptotic marker proteins. These results indicate that IDPc may play an important role in regulating the apoptosis induced by staurosporine and the sensitizing effect of IDPc siRNA on the apoptotic cell death of HeLa cells offers the possibility of developing a modifier of cancer chemotherapy.
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http://dx.doi.org/10.1080/10715760802653661DOI Listing
February 2009

Induction of sulfiredoxin via an Nrf2-dependent pathway and hyperoxidation of peroxiredoxin III in the lungs of mice exposed to hyperoxia.

Antioxid Redox Signal 2009 May;11(5):937-48

Division of Life and Pharmaceutical Sciences, Ewha Womans University, Seoul, Korea.

The cysteine residue at the active site of peroxiredoxin (Prx) I, Prx II, or Prx III is reversibly hyperoxidized to cysteine sulfinic acid, with concomitant loss of peroxidase activity, during normal catalysis. Sulfiredoxin (Srx) is the enzyme responsible for reversing this hyperoxidation. We now show that the expression of Srx at both the mRNA and protein levels is increased markedly in the lungs of mice exposed to hyperoxia. This hyperoxia-induced expression of Srx was not evident in mice deficient in the transcription factor Nrf2, indicating an essential role for an Nrf2 signaling pathway in this effect. Hyperoxia also elicited the accumulation of the sulfinic form of the mitochondrial enzyme Prx III, but not that of the cytosolic enzymes Prx I or Prx II, in lung tissue. This selective hyperoxidation of Prx III is likely due either to mitochondria being the major site of the hyperoxia-induced production of reactive oxygen species or to the translocation of Srx from the cytosol into mitochondria being rate limiting for the reduction of sulfinic Prx III. Hyperoxia induced the degradation of Prx III in Nrf2-deficient mice but not in wild-type animals, suggesting that, in the absence of a sufficient amount of Srx, sulfinic Prx III is converted to a form that is susceptible to proteolysis.
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http://dx.doi.org/10.1089/ars.2008.2325DOI Listing
May 2009

Glutathionylation regulates IkappaB.

Biochem Biophys Res Commun 2008 Aug 13;373(1):169-73. Epub 2008 Jun 13.

School of Life Sciences and Biotechnology, College of Natural Sciences, Kyungpook National University, Taegu 702-701, Republic of Korea.

Although there has been considerable interest in the regulation of NFkappaB activation by glutathionylation, the possibility of IkappaB as a target for glutathionylation has not been investigated. We now report that Cys(189) of IkappaB alpha is a target for S-glutathionylation. This modification is reversed by thiols such as dithiothreitol and GSH. The glutathionylated IkappaB alpha appears to be significantly less susceptible than is native protein to phosphorylation by IkappaB kinase and casein kinase II, as well as to in vitro ubiquitination. This finding suggests that glutathionylation plays a regulatory role, presumably through structural alterations. HeLa cells treated with oxidant inducing GSH oxidation such as diamide showed the accumulation of glutathionylated IkappaB alpha. This mechanism suggests an alternative modification to the redox regulation of cysteine in IkappaB alpha and a possible mechanism in the regulation of NFkappaB activation.
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http://dx.doi.org/10.1016/j.bbrc.2008.06.007DOI Listing
August 2008

Silencing of mitochondrial NADP+-dependent isocitrate dehydrogenase by small interfering RNA enhances heat shock-induced apoptosis.

Biochem Biophys Res Commun 2008 Feb 27;366(4):1012-8. Epub 2007 Dec 27.

School of Life Sciences and Biotechnology, College of Natural Sciences, Kyungpook National University, Taegu 702-701, Republic of Korea.

Heat shock may increase oxidative stress due to increased production of reactive oxygen species and/or the promotion of cellular oxidation events. Mitochondrial NADP(+)-dependent isocitrate dehydrogenase (IDPm) produces NADPH, an essential reducing equivalent for the antioxidant system. In this report, we demonstrate that silencing of IDPm expression in HeLa cells greatly enhances apoptosis induced by heat shock. Transfection of HeLa cells with an IDPm small interfering RNA (siRNA) markedly decreased activity of IDPm, enhancing the susceptibility of heat shock-induced apoptosis reflected by morphological evidence of apoptosis, DNA fragmentation, cellular redox status, mitochondria redox status and function, and the modulation of apoptotic marker proteins. These results indicate that IDPm may play an important role in regulating the apoptosis induced by heat shock and the sensitizing effect of IDPm siRNA on the apoptotic cell death of HeLa cells offers the possibility of developing a modifier of cancer therapy.
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http://dx.doi.org/10.1016/j.bbrc.2007.12.067DOI Listing
February 2008

Small interfering RNA-mediated silencing of mitochondrial NADP+-dependent isocitrate dehydrogenase enhances the sensitivity of HeLa cells toward tumor necrosis factor-alpha and anticancer drugs.

Free Radic Biol Med 2007 Oct 20;43(8):1197-207. Epub 2007 Jul 20.

School of Life Sciences and Biotechnology, College of Natural Sciences, Kyungpook National University, Taegu 702-701, Korea.

Tumor necrosis factor-alpha (TNF-alpha) and several anticancer drugs induce the production of reactive oxygen species, which play an important causative role in apoptotic cell death. Recently, we demonstrated that the control of mitochondrial redox balance and the cellular defense against oxidative damage is one of the primary functions of mitochondrial NADP(+)-dependent isocitrate dehydrogenase (IDPm) by supplying NADPH for antioxidant systems. In the present report, we show that silencing of IDPm expression in HeLa cells greatly enhances apoptosis induced by TNF-alpha and anticancer drugs. Transfection of HeLa cells with an IDPm small interfering RNA (siRNA) markedly decreased activity of IDPm, enhancing the susceptibility of anticancer agent-induced apoptosis reflected by morphological evidence of apoptosis, DNA fragmentation, cellular redox status, mitochondria redox status and function, and the modulation of apoptotic marker proteins. These results indicate that IDPm may play an important role in regulating the apoptosis induced by TNF-alpha and anticancer drugs and the sensitizing effect of IDPm siRNA on the apoptotic cell death of HeLa cells offers the possibility of developing a modifier of cancer chemotherapy.
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http://dx.doi.org/10.1016/j.freeradbiomed.2007.07.009DOI Listing
October 2007

Ursolic acid regulates high glucose-induced apoptosis.

Free Radic Res 2007 Jun;41(6):638-44

School of Life Sciences and Biotechnology, College of Natural Sciences, Kyungpook National University, Taegu, South Korea.

A high concentration of glucose has been implicated as a causal factor in initiation and progression of diabetic complications and there is evidence to suggest that hyperglycemia increases the production of free radicals and oxidative stress. Therefore, compounds that scavenge reactive oxygen species (ROS) may confer regulatory effects on high glucose-induced apoptosis. Ursolic acid (UA), a pentacyclic triterpene, is reported to have an antioxidant activity. We investigated the effect of UA on high glucose-induced apoptosis in U937 cells. Upon exposure to 35 mM glucose for two days, there was a distinct difference between untreated cells and cells pre-treated with 50 nM UA for 2 h in regard to cellular redox status and oxidative DNA damage to cells. UA pre-treated cells showed significant suppression of apoptotic features such as DNA fragmentation, damage to mitochondrial function and modulation of apoptotic marker proteins upon exposure to high glucose. This study indicates that UA may play an important role in regulating the apoptosis induced by high glucose presumably through scavenging of ROS.
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http://dx.doi.org/10.1080/10715760701227526DOI Listing
June 2007

Regulation of ionizing radiation-induced apoptosis by mitochondrial NADP+-dependent isocitrate dehydrogenase.

J Biol Chem 2007 May 9;282(18):13385-94. Epub 2007 Mar 9.

School of Life Sciences and Biotechnology, College of Natural Sciences, Kyungpook National University, Taegu 702-701, Korea.

Ionizing radiation induces the production of reactive oxygen species, which play an important causative role in apoptotic cell death. By supplying NADPH for antioxidant systems, we recently demonstrated that the control of mitochondrial redox balance and the cellular defense against oxidative damage are some of the primary functions of mitochondrial NADP(+)-dependent isocitrate dehydrogenase (IDPm). In this study, we demonstrate that modulation of IDPm activity in U937 cells regulates ionizing radiation-induced apoptosis. When we examined the regulatory role of IDPm against ionizing radiation-induced apoptosis in U937 cells transfected with the cDNA for mouse IDPm in sense and antisense orientations, a clear inverse relationship was observed between the amount of IDPm expressed in target cells and their susceptibility to apoptosis. Upon exposure to 2 gray gamma-irradiation, there was a distinct difference between the IDPm transfectant cells in regard to the morphological evidence of apoptosis, DNA fragmentation, cellular redox status, oxidative damage to cells, mitochondrial function, and the modulation of apoptotic marker proteins. In addition, transfection of HeLa cells with an IDPm small interfering RNA decreased the activity of IDPm, enhancing the susceptibility of radiation-induced apoptosis. Taken together, these results indicate that IDPm may play an important role in regulating the apoptosis induced by ionizing radiation, and the effect of IDPm small interfering RNA on HeLa cells offers the possibility of developing a modifier of radiation therapy.
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http://dx.doi.org/10.1074/jbc.M700303200DOI Listing
May 2007

Mitochondrial NADP+-dependent isocitrate dehydrogenase protects cadmium-induced apoptosis.

Mol Pharmacol 2006 Sep 19;70(3):1053-61. Epub 2006 Jun 19.

School of Life Sciences and Biotechnology, College of Natural Sciences, Kyungpook National University, Taegu 702-701, Korea.

Cadmium is known to exhibit high affinity for thiol groups and may therefore severely disturb many cellular functions. We have demonstrated that the control of mitochondrial redox balance and oxidative damage is one of the primary functions of mitochondrial NADP+-dependent isocitrate dehydrogenase (IDPm). When exposed to cadmium, IDPm was susceptible to loss of enzyme activity and structural alterations. Site-directed mutagenesis confirms that binding of cadmium occurs to a Cys379 of IDPm. We examined the antioxidant mechanism-mediated protective role of IDPm against cadmium-induced apoptosis with human embryonic kidney 293 cells transfected with the IDPm cDNA in sense and antisense orientations. As a result, we observed a clear inverse relationship between the amount of IDPm expressed in target cells and their susceptibility to cadmium-induced modulation of cellular redox status and apoptosis. In addition, loss of glutaredoxin (Grx, thioltransferase) activity by cadmium was more pronounced in antisense cells compared with the sense cells. When oxalomalate, a competitive inhibitor of IDPm, was administered to mice, inhibition of IDPm and Grx and enhanced susceptibility to apoptosis were observed upon their exposure to cadmium. These results suggest that IDPm plays an important protective role in cadmium-induced apoptosis by maintaining cellular redox status and by protection of Grx activity.
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http://dx.doi.org/10.1124/mol.106.023515DOI Listing
September 2006

Orchiectomy attenuates post-ischemic oxidative stress and ischemia/reperfusion injury in mice. A role for manganese superoxide dismutase.

J Biol Chem 2006 Jul 8;281(29):20349-56. Epub 2006 May 8.

Department of Anatomy, School of Medicine, Kyungpook National University, Daegu, Korea.

Males are much more susceptible to ischemia/reperfusion (I/R)-induced kidney injury when compared with females. Recently we reported that the presence of testosterone, rather than the absence of estrogen, plays a critical role in gender differences in kidney susceptibility to I/R injury in mice. Although reactive oxygen species and antioxidant defenses have been implicated in I/R injury, their roles remain to be defined. Here we report that the orchiectomized animal had significantly less lipid peroxidation and lower hydrogen peroxide levels in the kidney 4 and 24 h after 30 min of bilateral renal ischemia when compared with intact or dihydrotestosterone-treated orchiectomized males. The post-ischemic kidney expression and activity of manganese superoxide dismutase (MnSOD) in orchiectomized mice was much greater than in intact or dihydrotestosterone-administered orchiectomized mice. Four hours after 30 min of bilateral ischemia, superoxide formation was significantly lower in orchiectomized mice than in intact mice. In Madin-Darby canine kidney cells, a kidney epithelial cell line, 1 mm H(2)O(2) decreased MnSOD activity, an effect that was potentiated by pretreatment with dihydrotestosterone. Orchiectomy prevented the post-ischemic decrease of catalase activity. Treatment of male mice with manganese(III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP), a SOD mimetic, reduced the post-ischemic increase of plasma creatinine, lipid peroxidation, and tissue hydrogen peroxide. These results suggest that orchiectomy accelerates the post-ischemic activation of MnSOD and reduces reactive oxygen species and lipid peroxidation, resulting in reduced kidney susceptibility to I/R injury.
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http://dx.doi.org/10.1074/jbc.M512740200DOI Listing
July 2006

Regulation of replicative senescence by NADP+ -dependent isocitrate dehydrogenase.

Free Radic Biol Med 2006 Jan 11;40(1):110-9. Epub 2005 Oct 11.

School of Life Sciences and Biotechnology, College of Natural Sciences, Kyungpook National University, Taegu 702-701, Korea.

The free radical hypothesis of aging postulates that senescence is due to an accumulation of cellular oxidative damage, caused largely by reactive oxygen species that are produced as by-products of normal metabolic processes. Recently, we demonstrated that the control of cytosolic and mitochondrial redox balance and the cellular defense against oxidative damage is one of the primary functions of cytosolic (IDPc) and mitochondrial NADP+ -dependent isocitrate dehydrogenase (IDPm) by supplying NADPH for antioxidant systems. In this paper, we demonstrate that modulation of IDPc or IDPm activity in IMR-90 cells regulates cellular redox status and replicative senescence. When we examined the regulatory role of IDPc and IDPm against the aging process with IMR-90 cells transfected with cDNA for IDPc or IDPm in sense and antisense orientations, a clear inverse relationship was observed between the amount of IDPc or IDPm expressed in target cells and their susceptibility to senescence, which was reflected by changes in replicative potential, cell cycle, senescence-associated beta-galactosidase activity, expression of p21 and p53, and morphology of cells. Furthermore, lipid peroxidation, oxidative DNA damage, and intracellular peroxide generation were higher and cellular redox status shifted to a prooxidant condition in the cell lines expressing the lower level of IDPc or IDPm. The results suggest that IDPc and IDPm play an important regulatory role in cellular defense against oxidative stress and in the senescence of IMR-90 cells.
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http://dx.doi.org/10.1016/j.freeradbiomed.2005.08.021DOI Listing
January 2006

Regulation of mitochondrial NADP+-dependent isocitrate dehydrogenase activity by glutathionylation.

J Biol Chem 2005 Mar 14;280(11):10846-54. Epub 2005 Jan 14.

Department of Biochemistry, College of Natural Sciences, Kyungpook National University, Taegu 702-701, Korea.

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.
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http://dx.doi.org/10.1074/jbc.M411306200DOI Listing
March 2005