Inactivation of NADP(+)-dependent isocitrate dehydrogenase by reactive oxygen species

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 NADP(+)-dependent isocitrate dehydrogenase (ICDH) through supply of NADPH for antioxidant systems. When exposed to various reactive oxygen species such as hydrogen peroxide, singlet oxygen generated by photoactivated dye, superoxide anion, and hydroxyl radical produced by metal-catalyzed Fenton reactions, ICDH was susceptible to oxidative modification and damage, which was indicated by the loss of activity, fragmentation of the peptide as well as by the formation of carbonyl groups. Oxidative damage to ICDH was inhibited by antioxidant enzymes, free radical scavengers, and spin-trapping agents. The structural alterations of modified enzymes were indicated by the increase in thermal instability and binding of the hydrophobic probe 8-anilino-1-naphthalene sulfonic acid (ANSA). The reactive oxygen species-mediated damage to ICDH may result in the perturbation of cellular antioxidant defense mechanisms and subsequently lead to a pro-oxidant condition.     

Characterization of a low-activity allele of NADP+-dependent isocitrate dehydrogenase from Drosophila melanogaster

We have characterized biochemical effects of Idh ^GB1 in Drosophila melanogaster. This is a null-activity allele for NADP⁺-dependent isocitrate dehydrogenase (NADP-IDH) isolated from a natural population. The homozygous mutant strain has 5% of the NADP-IDH specific activity found in controls and less than 24% of the immunologically cross-reacting material (CRM). This mutation maps to 27.2 on the third chromosome, to the right of h. The biochemical phenotype of this mutant strain includes a coordinate reduction in malic enzyme (ME) specific activity and CRM and an increase in specific activity for the pentose-phosphate shunt enzymes, 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase. The K _m values for purified NADP-IDH are not different from those found for the purified control enzyme for NADP⁺ or isocitrate. It is suggested that this allele may represent a cis-acting control mutation for one of at least two loci involved in the production of NADP-IDH in D. melanogaster.Research supported by an Alberta Heritage Foundation for Medical Research Establishment Grant to MMB and a Natural Sciences and Engineering Research Council Operating Grant to JHW.     

Nad+-dependent isocitrate dehydrogenase from Arabidopsis thaliana. Characterization of two closely related subunits

Two cDNA clones which appear to encode different subunits of NAD⁺-dependent isocitrate dehydrogenase (IDH; EC 1.1.1.41) were identified by homology searches from the Arabidopsis EST database. These cDNA clones were obtained and sequenced; both encoded full-length messages and displayed 82.7% nucleotide sequence identity over the coding region. The deduced amino acid sequences revealed preprotein lengths of 367 residues, with an amino acid identity of 86.1%. Genomic Southern blot analysis showed distinct single-copy genes for both IDH subunits. Both IDH subunits were expressed as recombinant proteins in Escherichia coli, and polyclonal antibodies were raised to each subunit. The Arabidopsis cDNA clones were expressed in Saccharomyces cerevisiae mutants which were deficient in either one or both of the yeast NAD⁺-dependent IDH subunits. The Arabidopsis cDNA clones failed to complement the yeast mutations; although both IDH-I and IDH-II were expressed at detectable levels, neither protein was imported into the mitochondria.     

Cellular defense against UVB-induced phototoxicity by cytosolic NADP(+)-dependent isocitrate dehydrogenase

Ultraviolet (UV) radiation is known as a major cause of skin photoaging and photocarcinogenesis. Many harmful effects of UV radiation are associated with the generation of reactive oxygen species. Recently, we have shown that NADP(+)-dependent isocitrate dehydrogenase is involved in the supply of NADPH needed for GSH production against cellular oxidative damage. In this study we investigated the role of cytosolic form of NADP(+)-dependent isocitrate dehydrogenase (IDPc) against UV radiation-induced cytotoxicity by comparing the relative degree of cellular responses in three different NIH3T3 cells with stable transfection with the cDNA for mouse IDPc in sense and antisense orientations, where IDPc activities were 2.3-fold higher and 39% lower, respectively, than that in the parental cells carrying the vector alone. Upon exposure to UVB (312 nm), the cells with low levels of IDPc became more sensitive to cell killing. Lipid peroxidation, protein oxidation, oxidative DNA damage, and intracellular peroxide generation were higher in the cell-line expressing the lower level of IDPc. However, the cells with the highly overexpressed IDPc exhibited enhanced resistance against UV radiation, compared to the control cells. The data indicate that IDPc plays an important role in cellular defense against UV radiation-induced oxidative injury.     

Shifted Cytosolic NADP+-Dependent Isocitrate Dehydrogenase on 2-D Gel in the Brain of Genetically Epileptic El Mice

We observed a spot on two-dimensional (2-D) gel in the epileptic mutant strain El mice with a similar molecular weight but with a different isoelectric point of approximately 0.2, compared with its mother strain ddY mice. The collected protein from the El mice was identified as cytosolic NADP⁺-dependent isocitrate dehydrogenase by internal amino acid sequencing. The enzyme is known to be maximally active during the development of the brain and to play an important role in NADPH production for fatty acids and cholesterol synthesis. In addition, alterations in cholesterol synthesis early in the development of the mammalian brain have been reported to lead to chronic epilepsy. The results in the present study therefore suggest that cytosolic NADP⁺-dependent isocitrate dehydrogenase might be involved in the epileptogenesis of the El mouse.     

Interplay between ferritin metabolism, reactive oxygen species and nitric oxide

 The biological relevance of each of the three inorganic species – iron, oxygen, and nitric oxide (NO) – is crucial. Moreover, their metabolic pathways cross each other and thus create a complex network of connections responsible for the regulation of many essential biological processes. The iron storage protein ferritin, one of the main regulators of iron homeostasis, influences oxygen and NO metabolism. Here, examples are given of the biological interactions of the ferritin molecule (ferritin iron and ferritin shell) with reactive oxygen species (ROS) and NO. The focus is the regulation of ferritin expression by ROS and NO. From these data, ferritin emerges as an important cytoprotective component of the cellular response to ROS and NO. Also, by its ability to alter the amount of intracellular "free" iron, ferritin may affect the metabolism of ROS and NO. It is proposed that this putative activity of ferritin may constitute a missing link in the regulatory loop between iron, ROS, and NO. Received: 2 January 1997 / Accepted: 9 June 1997     

Changes in NADP+-linked isocitrate dehydrogenase during tomato fruit ripening

The activity of NADP⁺-specific isocitrate dehydrogenase (NADP⁺-IDH, EC 1.1.1.42) was investigated during the ripening of tomato (Lycopersicon esculentum Mill.) fruit. In the breaker stage, NADP⁺-IDH activity declined but a substantial recovery was observed in the late ripening stages when most lycopene synthesis occurs. These changes resulted in higher NADP⁺-IDH activity and specific polypeptide abundance in ripe than in green fruit pericarp. Most of the enzyme corresponded to the predominant cytosolic isoform which was purified from both green and ripe fruits. Fruit NADP⁺-IDH seems to be a dimeric enzyme having a subunit size of 48 kDa. The K _m values of the enzymes from green and ripe pericarp for NADP⁺, isocitrate and Mg²⁺ were not significantly different. The similar molecular and kinetic properties and chromatographic behaviour of the enzymes from the two kinds of tissue strongly suggest that the ripening process is not accompanied by a change in isoenzyme complement. The increase in NADP⁺-IDH in the late stage of ripening also suggests that this enzyme is involved in the metabolism of C₆ organic acids and in glutamate accumulation in ripe tissues.     

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.     

NADPH recycling systems in oxidative stressed pea nodules: a key role for the NADP<Superscript>+</Superscript>-dependent isocitrate dehydrogenase

The symbiosis between legumes and rhizobia is characterised by the formation of dinitrogen-fixing root nodules. In natural conditions, nitrogen fixation is strongly impaired by abiotic stresses which generate over-production of reactive oxygen species. Since one of the nodule main antioxidant systems is the ascorbate–glutathione cycle, NADPH recycling that is involved in glutathione reduction is of great relevance under stress conditions. NADPH is mainly produced by glucose 6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (6PGDH; EC 1.1.1.44) from the oxidative pentose phosphate pathway, and also by NADP⁺-dependent isocitrate dehydrogenase (ICDH; EC 1.1.1.42). In this work, 10 μM paraquat (PQ) was applied to pea roots in order to determine the in vivo relationship between oxidative stress and the activity of the NADPH-generating enzymes in nodules. Whereas G6PDH and 6PGDH activities remained unchanged, a remarkable induction of ICDH gene expression and a dramatic increase of the ICDH activity was observed during the PQ treatment. These results support that ICDH has a key role in NADPH recycling under oxidative stress conditions in pea root nodules.     

K(+) channel activity and redox status are differentially required for JNK activation by UV and reactive oxygen species

Upon exposure to ultraviolet (UV) radiation, osmotic changes or the presence of reactive oxygen species (ROS) c-Jun N-terminal kinases (JNKs) are rapidly activated. Extensive studies have elucidated molecular components that mediate the activation of JNKs. However, it remains unclear whether activation of JNKs by various stress signals involves different pathways. Here we show that K(+) channel activity is involved in mediating apoptosis induced by UV but not by H(2)O(2) in myelocytic leukemic ML-1 cells. Specifically, JNKs were rapidly phosphorylated upon treatment of ML-1 cells with UV and H(2)O(2). UV-induced, but not H(2)O(2)-induced, JNK-1 phosphorylation was inhibited by pretreatment with 4-aminopyridine (4-AP), a K(+) channel blocker. 4-AP also blocked UV-induced increase in JNK activity as well as p38 phosphorylation. Immunofluorescent microscopy revealed that phosphorylated JNKs were concentrated at centrosomes in ML-1 cells and that these proteins underwent rapid subcellular translocation upon UV treatment. Consistently, the subcellular translocation of JNKs induced by UV was largely blocked by 4-AP. Furthermore, UV-induced JNK activation was blocked by NEM, a sulfhydryl alkylating agent also affecting K(+) current. Both UV- and H(2)O(2)-induced JNK activities were inhibited by glutathione, suggesting that the redox status does play an important role in the activation of JNKs. Taken together, our findings suggest that JNK activation by UV and H(2)O(2) is mediated by distinct yet overlapping pathways and that K(+) channel activity and redox status are differentially required for UV- and H(2)O(2)-induced activation of JNKs.     

Overexpression of the NADP+-specific isocitrate dehydrogenase gene (icdA) in citric acid-producing Aspergillus niger WU-2223L

In the tricarboxylic acid (TCA) cycle, NADP⁺-specific isocitrate dehydrogenase (NADP⁺-ICDH) catalyzes oxidative decarboxylation of isocitric acid to form α-ketoglutaric acid with NADP⁺ as a cofactor. We constructed an NADP⁺-ICDH gene (icdA)-overexpressing strain (OPI-1) using Aspergillus niger WU-2223L as a host and examined the effects of increase in NADP⁺-ICDH activity on citric acid production. Under citric acid-producing conditions with glucose as the carbon source, the amounts of citric acid produced and glucose consumed by OPI-1 for the 12-d cultivation period decreased by 18.7 and 10.5%, respectively, compared with those by WU-2223L. These results indicate that the amount of citric acid produced by A. niger can be altered with the NADP⁺-ICDH activity. Therefore, NADP⁺-ICDH is an important regulator of citric acid production in the TCA cycle of A. niger. Thus, we propose that the icdA gene is a potentially valuable tool for modulating citric acid production by metabolic engineering.     

NADP+-dependent acetaldehyde dehydrogenase from Zymomonas mobilis

An NADP⁺-linked acetaldehyde dehydrogenase (EC 1.2.1.4) from the ethanol producing bacterium Zymomonas mobilis was purified 180-fold to homogeneity. The enzyme is a cytosolic protein with an isoelectric point of 8.0 and has an apparent molecular weight of 210000. It showed a single band in sodium dodecylsulfate gel electrophoresis with a molecular weight of 55000, which indicates that it consists of four probably identical subunits. The apparent K _m values for the substrate acetaldehyde were 57 M and for the cosubstrate NADP⁺ 579 M. The enzyme was almost inactive with NAD⁺ as cofactor. Several other aldehydes besides acetaldehyde were accepted as a substrate but not formaldehyde or trichloroacetaldehyde. In anaerobically grown cells of Zymomonas mobilis the enzyme showed a specific activity of 0.035 U/mg protein but its specific activity could be increased up to 0.132 U/mg protein by adding acetaldehyde to the medium during the exponential growth phase or up to 0.284 U/mg protein when cells were grown under aeration. The physiological role of the enzyme is discussed.Abbreviations ALD-DH acetaldehyde dehydrogenases from Z. mobilis - DTT dithiothreitol - MES 2-(N-morpholino)ethanesulfonic acid - MOPS 3-(N-morpholino)propanesulfonic acid - SDS sodium dodecylsulfate Dedicated to Prof. Dr. H.-G. Schlegel, Universität Göttingen, on the occasion of his 65th birthday     

Crystal structure studies of NADP dependent isocitrate dehydrogenase from Thermus thermophilus exhibiting a novel terminal domain

NADP⁺ dependent isocitrate dehydrogenase (IDH) is an enzyme catalyzing oxidative decarboxylation of isocitrate into oxalosuccinate (intermediate) and finally the product α-ketoglutarate. The crystal structure of Thermus thermophilus isocitrate dehydrogenase (TtIDH) ternary complex with citrate and cofactor NADP⁺ was determined using X-ray diffraction method to a resolution of 1.80 Å. The overall fold of this protein was resolved into large domain, small domain and a clasp domain. The monomeric structure reveals a novel terminal domain involved in dimerization, very unique and novel domain when compared to other IDH’s. And, small domain and clasp domain showing significant differences when compared to other IDH’s of the same sub-family. The structure of TtIDH reveals the absence of helix at the clasp domain, which is mainly involved in oligomerization in other IDH’s. Also, helices/beta sheets are absent in the small domain, when compared to other IDH’s of the same sub family. The overall TtIDH structure exhibits closed conformation with catalytic triad residues, Tyr144-Asp248-Lys191 are conserved. Oligomerization of the protein is quantized using interface area and subunit–subunit interactions between protomers. Overall, the TtIDH structure with novel terminal domain may be categorized as a first structure of subfamily of type IV.     

Age-dependent change in reactive oxygen species and nitric oxide generation by rat alveolar macrophages

Immunosenescence is an age-associated dysregulation of the immune function, which contributes to increased susceptibility to disease in the elderly. Alveolar macrophages (AM) are known phagocytes that generate reactive oxygen species (ROS) and nitric oxide (NO), essential mediators for host defence. We studied phagocytosis, ROS and NO production in AM obtained from young, adult and senescent rats (1-2, 9-12 and 18-24 months old, respectively) after exposure to lipopolysaccharide (LPS, 0.1-10 microg mL(-1)), 12-O-tetradecanoylphorbol 13-acetate (TPA, 0.1 microg mL(-1)) or LPS + TPA in culture. Phagocytosis was significantly lower in control AM from adult rats than in AM from young animals. Nevertheless, AM from adult animals pretreated with LPS exhibited higher phagocytic capacity than AM from younger animals. ROS was identified by the NBT test at single cell level and quantified by automated image analysis. When TPA was added to all three populations, AM from adult and senescent animals responded more than AM from young animals. All LPS-stimulated AM produce more NO than controls. However, NO production increased three-, four- and two-fold in young, adult and senescent animals, respectively. Our results demonstrate that AM from young, adult and senescent animals display differential responsiveness to inflammatory mediators. Therefore, aging processes markedly affect AM metabolic functions and may further compromise the lung immune defence response, increasing adverse long-term health effects.     

Involvement of Ca in globular adiponectin-induced reactive oxygen species

Globular adiponectin (gAd) induces the generation of reactive oxygen species (ROS) and nitric oxide (NO) in the murine macrophage cell line RAW 264. We investigated the role of Ca²⁺ in gAd-induced ROS and NO generation. Pretreatment with BAPTA-AM, a selective chelator of intracellular Ca²⁺ ([Ca²⁺]_i), partially reduced gAd-induced generation of ROS and NO in gAd-treated RAW 264 cells. The lowest [Ca²⁺]_i occurred 30 min after gAd treatment, after which [Ca²⁺]_i increased continually and exceeded the initial level. The mitochondrial Ca²⁺ ([Ca²⁺]_m) detected by Rhod-2 fluorescence started to increase at 6 h after gAd treatment. Pretreatment with a NAD(P)H oxidase inhibitor, diphenyleneiodonium, prevented the reduction of [Ca²⁺]_i in the early phase after gAd treatment. Calcium depletion by BAPTA-AM had no effect on the gAd-induced [Ca²⁺]_m oscillation. The administration of a specific calmodulin inhibitor, calmidazolium, significantly suppressed gAd-induced ROS and NO generation and NOS activity.     

Oxalomalate,a competitive inhibitor of NADP+-dependent isocitrate dehydrogenase,enhances lipid peroxidation-mediated oxidative damage in U937 cells

Membrane lipid peroxidation processes yield products that may react with DNA and proteins to cause oxidative modifications. Cytosolic NADP+-dependent isocitrate dehydrogenase (ICDH) in U937 cells produces NADPH, an essential reducing equivalent for the antioxidant system. The protective role of ICDH against lipid peroxidation-mediated oxidative damage in U937 cells was investigated in control cells pre-treated with oxalomalate, a competitive inhibitor of ICDH. Upon exposure to 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH) to U937 cells, which induces lipid peroxidation in membranes, the viability was lower and the protein oxidation, lipid peroxidation, and oxidative DNA damage, reflected by an increase in 8-hydroxy-2'-deoxyguanosine, were higher in oxalomalate-treated cells as compared to control cells. We also observed the significant increase in the endogenous production of reactive oxygen species, as measured by the oxidation of 2',7'-dichlorodihydrofluorescin, as well as the significant decrease in the intracellular GSH level in oxalomalate-treated U937 cells upon exposure to AAPH. These results suggest that ICDH plays an important role as an antioxidant enzyme in cellular defense against lipid peroxidation-mediated oxidative damage through the removal of reactive oxygen species.     

Dandelion (Taraxacum officinale) flower extract suppresses both reactive oxygen species and nitric oxide and prevents lipid oxidation in vitro

Flavonoids and coumaric acid derivatives were identified from dandelion flower (Taraxacum officinale). Characteristics of chain-breaking antioxidants, such as extended lag phase and reduced propagation rate, were observed in oxidation of linoleic acid emulsion with the addition of dandelion flower extract (DFE). DFE suppressed both superoxide and hydroxyl radical, while the latter was further distinguished by both site-specific and non-specific hydroxyl radical inhibition. DPPH-radical-scavenging activity and a synergistic effect with alpha-tocopherol were attributed to the reducing activity derived from phenolic content of DFE. A significant (p < 0.05) and concentration-dependent, reduced nitric oxide production from acterial-lipopolysaccharide-stimulated mouse macrophage RAW264.7 cells was observed with the addition of DFE. Moreover, peroxyl-radical-induced intracellular oxidation of RAW264.7 cells was inhibited significantly (p < 0.05) by the addition of DFE over a range of concentrations. These results showed that the DFE possessed marked antioxidant activity in both biological and chemical models. Furthermore, the efficacy of DFE in inhibiting both reactive oxygen species and nitric oxide were attributed to its phenolic content.     

Modulation of mitochondrial K permeability and reactive oxygen species production by the p13 protein of human T-cell leukemia virus type 1

Human T-cell leukemia virus type-1 (HTLV-1) expresses an 87-amino acid protein named p13 that is targeted to the inner mitochondrial membrane. Previous studies showed that a synthetic peptide spanning an alpha helical domain of p13 alters mitochondrial membrane permeability to cations, resulting in swelling. The present study examined the effects of full-length p13 on isolated, energized mitochondria. Results demonstrated that p13 triggers an inward K⁺ current that leads to mitochondrial swelling and confers a crescent-like morphology distinct from that caused by opening of the permeability transition pore. p13 also induces depolarization, with a matching increase in respiratory chain activity, and augments production of reactive oxygen species (ROS). These effects require an intact alpha helical domain and strictly depend on the presence of K⁺ in the assay medium. The effects of p13 on ROS are mimicked by the K⁺ ionophore valinomycin, while the protonophore FCCP decreases ROS, indicating that depolarization induced by K⁺ vs. H⁺ currents has different effects on mitochondrial ROS production, possibly because of their opposite effects on matrix pH (alkalinization and acidification, respectively). The downstream consequences of p13-induced mitochondrial K⁺ permeability are likely to have an important influence on the redox state and turnover of HTLV-1-infected cells.     

Epithelial,dendritic, and CD4 T cell regulation of and by reactive oxygen and nitrogen species in allergic sensitization

Background

While many of the contributing cell types and mediators of allergic asthma are known, less well understood are the factors that induce allergy in the first place. Amongst the mediators speculated to affect initial allergen sensitization and the development of pathogenic allergic responses to innocuous inhaled antigens and allergens are exogenously or endogenously generated reactive oxygen species (ROS) and reactive nitrogen species (RNS).

Scope of review

The interactions between ROS/RNS, dendritic cells (DCs), and CD4⁺ T cells, as well as their modulation by lung epithelium, are of critical importance for the genesis of allergies that later manifest in allergic asthma. Therefore, this review will primarily focus on the initiation of pulmonary allergies and the role that ROS/RNS may play in the steps therein, using examples from our own work on the roles of NO₂ exposure and airway epithelial NF-κB activation.

Major conclusions

Endogenously generated ROS/RNS and those encountered from environmental sources interact with epithelium, DCs, and CD4⁺ T cells to orchestrate allergic sensitization through modulation of the activities of each of these cell types, which quantitiatively and qualitatively dictate the degree and type of the allergic asthma phenotype.

General significance

Knowledge of the effects of ROS/RNS at the molecular and cellular levels has the potential to provide powerful insight into the balance between inhalational tolerance (the typical immunologic response to an innocuous inhaled antigen) and allergy, as well as to potentially provide mechanistic targets for the prevention and treatment of asthma.