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.     

Inactivation of mitochondrial NADP+-dependent isocitrate dehydrogenase by hypochlorous acid

Myeoloperoxidase catalyses the formation of hypochlorous acid (HOCl) via reaction of H(2)O(2) with Cl(-) ion. Although HOCl is known to play a major role in the human immune system by killing bacteria and other invading pathogens, excessive generation of this oxidant is known to cause damage to tissue. Recently, it was demonstrated that the control of mitochondrial redox balance and oxidative damage is one of the primary functions of mitochondrial NADP(+)-dependent isocitrate dehydrogenase (IDPm) to supply NADPH for antioxidant systems. This study investigated whether the IDPm would be a vulnerable target of HOCl as a purified enzyme and in intact cells. Loss of enzyme activity was observed and the inactivation of IDPm was reversed by thiols. Transfection of HeLa cells with an IDPm small interfering RNA (siRNA) markedly enhanced HOCl-induced oxidative damage to cells. The HOCl-mediated damage to IDPm may result in the perturbation of the cellular antioxidant defense mechanisms and subsequently lead to a pro-oxidant condition.     

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

(−)-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.     

Regulation of high glucose-induced apoptosis by mitochondrial NADP+-dependent isocitrate dehydrogenase

A high concentration of glucose has been implicated as a causal factor in initiation and progression of diabetic kidney complications, and there is evidence to suggest that hyperglycemia increases the production of free radicals and oxidant stress. 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) to supply NADPH for antioxidant systems. In this report, we demonstrate that modulation of IDPm activity in HEK293 cells, an embryonic kidney cell line, regulates high glucose-induced apoptosis. When we examined the protective role of IDPm against high glucose-induced apoptosis with HEK293 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. The results suggest that IDPm plays an important protective role in apoptosis of HEK293 cells induced by a high concentration of glucose and may contribute to various pathologies associated with the long-term complications of diabetes.     

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

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.     

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

Abstract

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 mitochon-drial 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.     

Suppression of tumorigenesis in mitochondrial NADP+-dependent isocitrate dehydrogenase knock-out mice

The tumor host microenvironment is increasingly viewed as an important contributor to tumor growth and suppression. Cellular oxidative stress resulting from high levels of reactive oxygen species (ROS) contributes to various processes involved in the development and progress of malignant tumors including carcinogenesis, aberrant growth, metastasis, and angiogenesis. In this regard, the stroma induces oxidative stress in adjacent tumor cells, and this in turn causes several changes in tumor cells including modulation of the redox status, inhibition of cell proliferation, and induction of apoptotic or necrotic cell death. Because the levels of ROS are determined by a balance between ROS generation and ROS detoxification, disruption of this system will result in increased or decreased ROS level. Recently, we demonstrated that the control of mitochondrial redox balance and cellular defense against oxidative damage is one of the primary functions of mitochondrial NADP⁺-dependent isocitrate dehydrogenase (IDH2) that supplies NADPH for antioxidant systems. To explore the interactions between tumor cells and the host, we evaluated tumorigenesis between IDH2-deficient (knock-out) and wild-type mice in which B16F10 melanoma cells had been implanted. Suppression of B16F10 cell tumorigenesis was reproducibly observed in the IDH2-deficient mice along with significant elevation of oxidative stress in both the tumor and the stroma. In addition, the expression of angiogenesis markers was significantly down-regulated in both the tumor and the stroma of the IDH2-deficient mice. These results support the hypothesis that redox status-associated changes in the host environment of tumor-bearing mice may contribute to cancer progression.     

Modulation of NADP+-dependent isocitrate dehydrogenase in aging

Abstract

NADPH is an important cofactor in many biosynthesis pathways and the regeneration of reduced glutathione, critically important in cellular defense against oxidative damage. It is mainly produced by glucose-6-phosphate dehydrogenase, malic enzyme, and NADP⁺-specific isocitrate dehydrogenases (ICDHs). Here, we investigated age-related changes in ICDH activity and protein expression in IMR-90 human diploid fibroblast cells and tissues from Fischer 344 rats. We found that in IMR-90 cells the activity of cytosolic ICDH (IDPc) gradually increased with age up to the 46–48 population doubling level (PDL) and then gradually decreased at later PDL. 2′,7′-Dichloro-fluorescein fluorescence which reflects intracellular ROS generation was increased with aging in IMR-90 cells. In ad libitum-fed rats, we noted age-related, tissue-specific modulations of IDPc and mitochondrial ICDH (IDPm) activities and protein expression in the liver, kidney and testes. In contrast, ICDH activities and protein expression were not significantly modulated in diet-restricted rats. These data suggest that modulation of ICDH is an age-dependent and a tissue-specific phenomenon.     

Cellular defense against heat shock-induced oxidative damage by mitochondrial NADP+-dependent isocitrate dehydrogenase

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. The protective role of IDPm against heat shock in HEK293 cells, an embryonic kidney cell line, was investigated in control and cells transfected with the cDNA for IDPm, where IDPm activity was 6–7 fold higher than that in the control cells carrying the vector alone. Upon exposure to heat shock, the viability was lower and the protein oxidation, lipid peroxidation and oxidative DNA damage were higher in control cells as compared to HEK293 cells in which IDPm was over-expressed. We also observed the significant difference in the cellular redox status reflected by the endogenous production of reactive oxygen species, NADPH pool and GSH recycling between two cells. The results suggest that IDPm plays an important role as an antioxidant defense enzyme in cellular defense against heat shock through the removal of reactive oxygen species.     

Regulation of replicative senescence by NADP+ -dependent isocitrate dehydrogenase

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.     

Cellular defense against heat shock-induced oxidative damage by mitochondrial NADP+ -dependent isocitrate dehydrogenase

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. The protective role of IDPm against heat shock in HEK293 cells, an embryonic kidney cell line, was investigated in control and cells transfected with the cDNA for IDPm, where IDPm activity was 6-7 fold higher than that in the control cells carrying the vector alone. Upon exposure to heat shock, the viability was lower and the protein oxidation, lipid peroxidation and oxidative DNA damage were higher in control cells as compared to HEK293 cells in which IDPm was over-expressed. We also observed the significant difference in the cellular redox status reflected by the endogenous production of reactive oxygen species, NADPH pool and GSH recycling between two cells. The results suggest that IDPm plays an important role as an antioxidant defense enzyme in cellular defense against heat shock through the removal of reactive oxygen species.     

Catalytic mechanism of NADP(+)-dependent isocitrate dehydrogenase: implications from the structures of magnesium-isocitrate and NADP+ complexes

The structures of NADP+ and magnesium isocitrate bound to the NADP(+)-dependent isocitrate dehydrogenase of Escherichia coli have been determined and refined at 2.5-A resolution. NADP+ is bound by the large domain of isocitrate dehydrogenase, a structure that has little similarity to the supersecondary structure of the nucleotide-binding domain of the lactate dehydrogenase-like family of nucleotide-binding proteins. The coenzyme-binding site confirms the fundamentally different evolution of the isocitrate dehydrogenase-like and the lactate dehydrogenase-like classes of nucleotide-binding proteins. In the magnesium-isocitrate complex, magnesium is coordinated to the alpha-carboxylate and alpha-hydroxyl oxygen of isocitrate in a manner suitable for stabilization of a negative charge on the hydroxyl oxygen during both the dehydrogenation and decarboxylation steps of the conversion of isocitrate to alpha-ketoglutarate. The metal ion is also coordinated by aspartate side chains 283' (of the second subunit of the dimer) and 307 and two water molecules in a roughly octahedral arrangement. On the basis of the geometry of the active site, the base functioning in the dehydrogenation step is most likely aspartate 283'. E. coli isocitrate dehydrogenase transfers a hydride stereospecifically to the A-side of NADP+, and models for a reactive ternary complex consistent with this stereospecificity are discussed.     

Control of mitochondrial redox balance and cellular defense against oxidative damage by mitochondrial NADP+-dependent isocitrate dehydrogenase

Mitochondria are the major organelles that produce reactive oxygen species (ROS) and the main target of ROS-induced damage as observed in various pathological states including aging. Production of NADPH required for the regeneration of glutathione in the mitochondria is critical for scavenging mitochondrial ROS through glutathione reductase and peroxidase systems. We investigated the role of mitochondrial NADP(+)-dependent isocitrate dehydrogenase (IDPm) in controlling the mitochondrial redox balance and subsequent cellular defense against oxidative damage. We demonstrate in this report that IDPm is induced by ROS and that decreased expression of IDPm markedly elevates the ROS generation, DNA fragmentation, lipid peroxidation, and concurrent mitochondrial damage with a significant reduction in ATP level. Conversely, overproduction of IDPm protein efficiently protected the cells from ROS-induced damage. The protective role of IDPm against oxidative damage may be attributed to increased levels of a reducing equivalent, NADPH, needed for regeneration of glutathione in the mitochondria. Our results strongly indicate that IDPm is a major NADPH producer in the mitochondria and thus plays a key role in cellular defense against oxidative stress-induced damage.     

Inactivation of NADP+-dependent isocitrate dehydrogenase by lipid peroxidation products

Membrane lipid peroxidation processes yield products that may react with proteins to cause oxidative modification. 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) through to supply NADPH for antioxidant systems. When exposed to lipid peroxidation products, such as malondialdehyde (MDA), 4-hydroxynonenal (HNE) and lipid hydroperoxide, ICDH was susceptible to oxidative damage, which was indicated by the loss of activity and the formation of carbonyl groups. The structural alterations of modified enzymes were indicated by the change in thermal stability, intrinsic tryptophan fluorescence and binding of the hydrophobic probe 8-anilino 1-napthalene sulfonic acid. Upon exposure to 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH), which induces lipid peroxidation in membrane, a significant decrease in both cytosolic and mitochondrial ICDH activities were observed in U937 cells. Using immunoprecipitation and immunoblotting, we were able to isolate and positively identify HNE adduct in mitochondrial ICDH from AAPH-treated U937 cells. The lipid peroxidation-mediated damage to ICDH may result in the perturbation of the cellular antioxidant defense mechanisms and subsequently lead to a pro-oxidant condition.     

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

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.     

Crystal structure of NAD-dependent malate dehydrogenase complexed with NADP(H)

For better understanding of the coenzyme specificity in NAD-dependent MDH (tMDH) from Thermus flavus AT-62, we determined the crystal structures of tMDH-NADP(H) complex at maximally 1.65 A resolution. The overall structure is almost the same as that of the tMDH-NADH complex. However, NADP(H) binds to tMDH in the reverse orientation, where adenine occupies the position near the catalytic center and nicotinamide is positioned at the adenine binding site of the tMDH-NADH complex. Consistent with this, kinetic analysis of the malate-oxidizing reaction revealed that NADP(+) inhibited tMDH at high concentrations. This has provided the first evidence for the alternative binding mode of the nicotinamide coenzyme, that has pseudo-symmetry in its structure, in a single enzyme.     

The distribution pattern of NADP+-dependent isocitrate dehydrogenase in rat liver

Activities of the NADP+-dependent isocitrate dehydrogenase were measured along the entire sinusoidal path (1) between small portal tracts and central veins and (2) between regions of adjoining septal branches and central veins in the liver of male Wistar rats using a Lowry technique. The measured activities show a slight increase from the periportal to the perivenous end, whereas no such septal-) perivenous gradient could be established. These profiles of enzyme activity give further support to previous studies, suggesting functional heterogeneity of liver sinusoids and their abutting hepatocytes related to morphological differences of the sinusoidal bed.     

Thermosensitive phenotype of Escherichia coli mutant lacking NADP+-dependent isocitrate dehydrogenase

Heat shock may increase oxidative stress due to increased production of reactive oxygen species and/or the promotion of cellular oxidation events. NADP(+)-dependent isocitrate dehydrogenase (ICDH) in Escherichia coli produces NADPH, an essential reducing equivalent for the antioxidant system. The protective role of ICDH against heat shock in E. coli was investigated in wild-type and ICDH-deficient strains. Upon exposure to heat shock, the viability was lower and the protein oxidation was higher in mutant cells as compared to wild-type cells. Induction and inactivation of antioxidant enzymes were observed after their exposure to heat shock both in wild-type and in mutant cells. However, wild-type cells maintained significantly higher activities of antioxidant enzymes than did mutant cells. These results suggest that ICDH plays an important role as an antioxidant enzyme in cellular defense against heat shock through the removal of reactive oxygen species as well as in the protection of other antioxidant enzymes.     

Ligands of the Mn2+ bound to porcine mitochondrial NADP-dependent isocitrate dehydrogenase, as assessed by mutagenesis

Pig heart mitochondrial NADP-dependent isocitrate dehydrogenase requires a divalent metal ion for catalysis, and metal-isocitrate is its preferred substrate. On the basis of the crystal structure of the enzyme-Mn(2+)-isocitrate complex, Asp(252), Asp(275), and Asp(279) were selected as targets for site-directed mutagenesis to evaluate the roles of these residues as ligands of the metal ion. The circular dichroism spectra of the purified mutant enzymes are similar to that of wild-type enzyme indicating there are no appreciable conformational changes. The K(m) values for isocitrate and for Mn(2+) are increased in the asparagine and histidine mutants at positions 252 and 275; while for cysteine mutants at the same positions, the K(m)'s are not changed appreciably. Mutants at position 279 exhibit only a small change in K(m) for isocitrate. These results indicate that Asp(252) and Asp(275) are ligands of enzyme-bound Mn(2+)and influence the binding of Mn(2+)-isocitrate. Cysteine is an acceptable substitute for aspartate as a ligand of Mn(2+). The pK(aes)'s of D252C and D275C enzymes are similar to that of the wild-type enzyme (about 5.2), while the pK(aes) of D279C is a little lower (about 4.7). These findings suggest that the V(max)'s of the D252C, D275C, and D279C enzymes depend on the ionizable form of the same group as in wild-type enzyme and neither Asp(252), Asp(275), nor Asp(279) acts as the general base in the enzymatic reaction. For wild-type enzyme, the pK(aes) varies with the metal ion used with Mg(2+) > Cd(2+) > Mn(2+) > Co(2+), similar to the order of the pK's for these four metal-bound waters. We therefore attribute the pH dependence of V(max) to the deprotonation of the metal-coordinated hydroxyl group of isocitrate bound to isocitrate dehydrogenase.     

Crystal structure of chicken liver dihydrofolate reductase complexed with NADP+ and biopterin.

The 2.2-A crystal structure of chicken liver dihydrofolate reductase (EC 1.5.1.3, DHFR) has been solved as a ternary complex with NADP+ and biopterin (a poor substrate). The space group and unit cell are isomorphous with the previously reported structure of chicken liver DHFR complexed with NADPH and phenyltriazine [Volz, K. W., Matthews, D. A., Alden, R. A., Freer, S. T., Hansch, C., Kaufman, B. T., & Kraut, J. (1982) J. Biol. Chem. 257, 2528-2536]. The structure contains an ordered water molecule hydrogen-bonded to both hydroxyls of the biopterin dihydroxypropyl group as well as to O4 and N5 of the biopterin pteridine ring. This water molecule, not observed in previously determined DHFR structures, is positioned to complete a proposed route for proton transfer from the side-chain carboxylate of E30 to N5 of the pteridine ring. Protonation of N5 is believed to occur during the reduction of dihydropteridine substrates. The positions of the NADP+ nicotinamide and biopterin pteridine rings are quite similar to the nicotinamide and pteridine ring positions in the Escherichia coli DHFR.NADP+.folate complex [Bystroff, C., Oatley, S. J., & Kraut, J. (1990) Biochemistry 29, 3263-3277], suggesting that the reduction of biopterin and the reduction of folate occur via similar mechanisms, that the binding geometry of the nicotinamide and pteridine rings is conserved between DHFR species, and that the p-aminobenzoylglutamate moiety of folate is not required for correct positioning of the pteridine ring in ground-state ternary complexes. Instead, binding of the p-aminobenzoylglutamate moiety of folate may induce the side chain of residue 31 (tyrosine or phenylalanine) in vertebrate DHFRs to adopt a conformation in which the opening to the pteridine binding site is too narrow to allow the substrate to diffuse away rapidly. A reverse conformational change of residue 31 is proposed to be required for tetrahydrofolate release.