Glutamate Cysteine Ligase Activity Research Articles

Enhanced glutathione biosynthetic capacity promotes resistance to As3+-induced apoptosis

Trivalent arsenite (As3+) is a known human carcinogen capable of inducing both cellular transformation and apoptotic cell death by mechanisms involving the production of reactive oxygen species. The tripeptide antioxidant glutathione (GSH) constitutes a vital cellular defense mechanism against oxidative stress. While intracellular levels of GSH are an important determinant of cellular susceptibility to undergo apoptotic cell death, it is not known whether cellular GSH biosynthetic capacity per se regulates As3+-induced apoptosis. The rate-limiting enzyme in GSH biosynthesis is glutamate cysteine ligase (GCL), a heterodimeric holoenzyme composed of a catalytic (GCLC) and a modifier (GCLM) subunit. To determine whether increased GSH biosynthetic capacity enhanced cellular resistance to As3+-induced apoptotic cell death, we utilized a mouse liver hepatoma (Hepa-1c1c7) cell line stably overexpressing both GCLC and GCLM. Overexpression of the GCL subunits increased GCL holoenzyme formation and activity and inhibited As3+-induced apoptosis. This cytoprotective effect was associated with a decrease in As3+-induced caspase activation, cleavage of caspase substrates and translocation of cytochrome c to the cytoplasm. In aggregate, these findings demonstrate that enhanced GSH biosynthetic capacity promotes resistance to As3+-induced apoptosis by preventing mitochondrial dysfunction and cytochrome c release and highlight the role of the GSH antioxidant defense system in dictating hepatocyte sensitivity to As3+-induced apoptotic cell death.

Manipulation of cellular GSH biosynthetic capacity via TAT-mediated protein transduction of wild-type or a dominant-negative mutant of glutamate cysteine ligase alters cell sensitivity to oxidant-induced cytotoxicity

The glutathione (GSH) antioxidant defense system plays a central role in protecting mammalian cells against oxidative injury. Glutamate cysteine ligase (GCL) is the rate-limiting enzyme in GSH biosynthesis and is a heterodimeric holoenzyme composed of catalytic (GCLC) and modifier (GCLM) subunits. As a means of assessing the cytoprotective effects of enhanced GSH biosynthetic capacity, we have developed a protein transduction approach whereby recombinant GCL protein can be rapidly and directly transferred into cells when coupled to the HIV TAT protein transduction domain. Bacterial expression vectors encoding TAT fusion proteins of both GCL subunits were generated and recombinant fusion proteins were synthesized and purified to near homogeneity. The TAT-GCL fusion proteins were capable of heterodimerization and formation of functional GCL holoenzyme in vitro. Exposure of Hepa-1c1c7 cells to the TAT-GCL fusion proteins resulted in the time- and dose-dependent transduction of both GCL subunits and increased cellular GCL activity and GSH levels. A heterodimerization-competent, enzymatically deficient GCLC-TAT mutant was also generated in an attempt to create a dominant-negative suppressor of GCL. Transduction of cells with a catalytically inactive GCLC(E103A)-TAT mutant decreased cellular GCL activity in a dose-dependent manner. TAT-mediated manipulation of cellular GCL activity was also functionally relevant as transduction with wild-type GCLC(WT)-TAT or mutant GCLC(E103A)-TAT conferred protection or enhanced sensitivity to H 2O 2-induced cell death, respectively. These findings demonstrate that TAT-mediated transduction of wild-type or dominant-inhibitory mutants of the GCL subunits is a viable means of manipulating cellular GCL activity to assess the effects of altered GSH biosynthetic capacity.

Mechanistic Details of Glutathione Biosynthesis Revealed by Crystal Structures of Saccharomyces cerevisiae Glutamate Cysteine Ligase

Glutathione is a thiol-disulfide exchange peptide critical for buffering oxidative or chemical stress, and an essential cofactor in several biosynthesis and detoxification pathways. The rate-limiting step in its de novo biosynthesis is catalyzed by glutamate cysteine ligase, a broadly expressed enzyme for which limited structural information is available in higher eukaryotic species. Structural data are critical to the understanding of clinical glutathione deficiency, as well as rational design of enzyme modulators that could impact human disease progression. Here, we have determined the structures of Saccharomyces cerevisiae glutamate cysteine ligase (ScGCL) in the presence of glutamate and MgCl(2) (2.1 A; R = 18.2%, R(free) = 21.9%), and in complex with glutamate, MgCl(2), and ADP (2.7 A; R = 19.0%, R(free) = 24.2%). Inspection of these structures reveals an unusual binding pocket for the alpha-carboxylate of the glutamate substrate and an ATP-independent Mg(2+) coordination site, clarifying the Mg(2+) dependence of the enzymatic reaction. The ScGCL structures were further used to generate a credible homology model of the catalytic subunit of human glutamate cysteine ligase (hGCLC). Examination of the hGCLC model suggests that post-translational modifications of cysteine residues may be involved in the regulation of enzymatic activity, and elucidates the molecular basis of glutathione deficiency associated with patient hGCLC mutations.

Effect of melatonin in the antioxidant defense system in the locomotor muscles of the estuarine crab Neohelice granulata (Decapoda, Brachyura)

In vertebrates, many studies verified different effects of melatonin in the antioxidant defense system (ADS). In crustaceans, few studies have been conducted to verify this possibility. We verified the melatonin effects in the crab Neohelice granulata using low (0.002 and 0.02 pmol/crab) and high (2.0 and 20.0 pmol/crab) melatonin dosages in short-term (0.5 h) and long-term (9.5 h) experiments. We analyzed the antioxidant capacity against peroxyl radicals (ACAP), reactive oxygen species (ROS) concentration, levels of by products of lipid peroxidation (LPO), oxygen consumption (VO 2), the activity of glutamate cysteine ligase (γ-GCL) and catalase (CAT) and glutathione content (GSH). Finally, the effects of exogenous melatonin were verified in terms of melatonin and N 1-acetyl- N 2-formyl-5-methoxykynuramine (AFMK) content in the muscles of N. granulata. In short-term experiment and low dosages, melatonin increased the VO 2, γ-GCL activity and GSH content ( p < 0.05) and decreased melatonin content ( p < 0.05) without effects in ROS, ACAP and LPO ( p > 0.05). Possibly, melatonin is acting in the ADS increasing its efficiency and/or acting in mitochondrial activity and/or through signaling muscles to increase its consumption. AFMK was only detected in the eyestalk and cerebroid ganglia. In high dosages melatonin effects decreased, possibly by the desensitization of their receptors. In long-term experiment, melatonin decreased ACAP ( p < 0.05), and CAT activity ( p < 0.05) in low dosages. In high dosages melatonin reduced VO 2 ( p < 0.05) and increased ACAP ( p < 0.05), possibly stimulating others components of the ADS. In conclusion, melatonin in the locomotor muscles of N. granulata affects the antioxidant/pro-oxidant balance in a time and dosage dependent manner.

Adaptation to G93Asuperoxide dismutase 1 in a motor neuron cell line model of amyotrophic lateral sclerosis

Motor neuron degeneration in amyotrophic lateral sclerosis involves oxidative damage. Glutathione (GSH) is critical as an antioxidant and a redox modulator. We used a motor neuronal cell line (NSC-34) to investigate whether wild-type and familial amyotrophic lateral sclerosis-linked G93A mutant Cu,Zn superoxide dismutase (wt/G93ASOD1) modified the GSH pool and glutamate cysteine ligase (GCL), the rate-limiting enzyme for GSH synthesis. We studied the effect of various G93ASOD1 levels and exposure times. Mutant Cu,Zn superoxide dismutase induced an adaptive process involving the upregulation of GSH synthesis, even at very low expression levels. However, cells with a high level of G93ASOD1 cultured for 10 weeks showed GSH depletion and a decrease in expression of the modulatory subunit of GCL. These cells also had lower levels of GSH and GCL activity was not induced after treatment with the pro-oxidant tert-butylhydroquinone. Cells with a low level of G93ASOD1 maintained higher GSH levels and GCL activity, showing that the exposure time and the level of the mutant protein modulate GSH synthesis. We conclude that failure of the regulation of the GSH pathway caused by G93ASOD1 may contribute to motor neuron vulnerability and we identify this pathway as a target for therapeutic intervention.

Distinct Nrf1/2-independent mechanisms mediate As 3+-induced glutamate-cysteine ligase subunit gene expression in murine hepatocytes

Trivalent arsenite (As 3+) is a known human carcinogen that is also capable of inducing apoptotic cell death. Increased production of reactive oxygen species is thought to contribute to both the carcinogenic and the cytotoxic effects of As 3+. Glutathione (GSH) constitutes a vital cellular defense mechanism against oxidative stress. The rate-limiting enzyme in GSH biosynthesis is glutamate-cysteine ligase (GCL), a heterodimeric holoenzyme composed of a catalytic (GCLC) and a modifier (GCLM) subunit. In this study, we demonstrate that As 3+ coordinately upregulates Gclc and Gclm mRNA levels in a murine hepatocyte cell line resulting in increased GCL subunit protein expression, holoenzyme formation, and activity. As 3+ increased the rate of transcription of both the Gclm and the Gclc genes and induced the posttranscriptional stabilization of Gclm mRNA. The antioxidant N-acetylcysteine abolished As 3+-induced Gclc expression and attenuated induction of Gclm. As 3+ induction of Gclc and Gclm was also differentially regulated by the MAPK signaling pathways and occurred independent of the Nrf1/2 transcription factors. These findings demonstrate that distinct transcriptional and posttranscriptional mechanisms mediate the coordinate induction of the Gclc and Gclm subunits of GCL in response to As 3+ and highlight the potential importance of the GSH antioxidant defense system in regulating As 3+-induced responses in hepatocytes.

Curcumin, quercetin, and tBHQ modulate glutathione levels in astrocytes and neurons: importance of the glutamate cysteine ligase modifier subunit

A decrease in GSH levels, the main redox regulator, can be observed in neurodegenerative diseases as well as in schizophrenia. In search for substances able to increase GSH, we evaluated the ability of curcumin (polyphenol), quercetin (flavonoid), and tert-butylhydroquinone (tBHQ) to up-regulate GSH-synthesizing enzymes. The gene expression, activity, and product levels of these enzymes were measured in cultured neurons and astrocytes. In astrocytes, all substances increased GSH levels and the activity of the rate-limiting synthesizing enzyme, glutamate cysteine ligase (GCL). In neurons, curcumin and to a lesser extent tBHQ increased GCL activity and GSH levels, while quercetin decreased GSH and led to cell death. In the two cell types, the gene that showed the greatest increase in its expression was the one coding for the modifier subunit of GCL (GCLM). The increase in mRNA levels of GCLM was 3 to 7-fold higher than that of the catalytic subunit. In astrocytes from GCLM-knock-out mice showing low GSH (-80%) and low GCL activity (-50%), none of the substances succeeded in increasing GSH synthesis. Our results indicate that GCLM is essential for the up-regulation of GCL activity induced by curcumin, quercetin and tBHQ.

Effects of arsenic (As) exposure on the antioxidant status of gills of the zebrafish Danio rerio (Cyprinidae)

In fishes, arsenic (As) is absorbed via the gills and is capable of causing disturbance to the antioxidant system. The objective of present study was to evaluate antioxidant responses after As exposure in gills of zebrafish ( Danio rerio, Cyprinidae). Fish were exposed for 48 h to three concentration of As, including the highest As concentration allowed by current Brazilian legislation (10 μg As/L). A control group was exposed to tap water (pH 8.0; 26 °C; 7.20 mg O 2/L). As exposure resulted in (1) an increase ( p < 0.05) of glutathione (GSH) levels after exposure to 10 and 100 μg As/L, (2) an increase of the glutamate cysteine ligase (GCL) activity in the same concentrations ( p < 0.05), (3) no significant differences in terms of glutathione reductase, glutathione- S-transferase and catalase activities; (4) a significantly lower ( p < 0.05) oxygen consumption after exposure to 100 μg As/L; (4) no differences in terms of oxygen reactive species generation and lipid peroxidation content ( p > 0,05). In the gills, only inorganic As was detected. Overall, it can be concluded that As affected the antioxidant responses increasing GCL activity and GSH levels, even at concentration considered safe by Brazilian legislation.

Impaired Metabolomics of Sulfur-Containing Substances in Rats Acutely Treated with Carbon Tetrachloride.

Impairment of hepatic metabolism of sulfur-containing amino acids has been known to be linked with induction of liver injury. We determined the early changes in the transsulfuration reactions in liver of rats challenged with a toxic dose of CCl4 (2 mmol/kg, ip). Both hepatic methionine concentration and methionine adenosyltransferase activity were increased, but S-adenosylmethionine level did not change. Hepatic cysteine was increased significantly from 4 h after CCl4 treatment. Glutathione (GSH) concentration in liver was elevated in 4~8 h and then returned to normal in accordance with the changes in glutamate cysteine ligase activity. Cysteine dioxygenase activity and hypotaurine concentration were also elevated from 4 h after the treatment. However, plasma GSH concentration was increased progressively, reaching a level at least several fold greater than normal in 24 h. γ-Glutamyltransferase activity in kidney or liver was not altered by CCl4, suggesting that the increase in plasma GSH could not be attributed to a failure of GSH cycling. The results indicate that acute liver injury induced by CCl4 is accompanied with extensive alterations in the metabolomics of sulfur-containing amino acids and related substances. The major metabolites and products of the transsul-furation pathway, including methionine, cysteine, hypotaurine, and GSH, are all increased in liver and plasma. The physiological significance of the change in the metabolomics of sulfur-containing substances and its role in the induction of liver injury need to be explored in future studies.

Skin fibroblast model to study an impaired glutathione synthesis: Consequences of a genetic polymorphism on the proteome

An impaired glutathione (GSH) synthesis was observed in several multifactorial diseases, including schizophrenia and myocardial infarction. Genetic studies revealed an association between schizophrenia and a GAG trinucleotide repeat (TNR) polymorphism in the catalytic subunit (GCLC) of the glutamate cysteine ligase (GCL). Disease-associated genotypes of this polymorphism correlated with a decrease in GCLC protein expression, GCL activity and GSH content. To clarify consequences of a decreased GCL activity at the proteome level, three schizophrenia patients and three controls have been selected based on the GCLC GAG TNR polymorphism. Fibroblast cultures were obtained by skin biopsy and were challenged with tert-butylhydroquinone (t-BHQ), a substance known to induce oxidative stress. Proteome changes were analyzed by two dimensional gel electrophoresis (2-DE) and results revealed 10 spots that were upregulated in patients following t-BHQ treatment, but not in controls. Nine corresponding proteins could be identified by MALDI mass spectrometry and these proteins are involved in various cellular functions, including energy metabolism, oxidative stress response, and cytoskeletal reorganization. In conclusion, skin fibroblasts of subjects with an impaired GSH synthesis showed an altered proteome reaction in response to oxidative stress. Furthermore, the study corroborates the use of fibroblasts as an additional mean to study vulnerability factors of psychiatric diseases.

HIF1 Induced Glutathione Dysregulation in Platelets Associated with Increased Risk of Vascular Disease in Chuvash Polycythemia.

The von Hippel-Lindau protein-hypoxia-inducible factor pathway is a transcriptional system controlling cellular responses to hypoxia. Hypoxia-inducible factor-1 (HIF-1) is a heterodimer of α and β subunits. Under normoxia, the prolyl residues of the α subunit are hydroxylated allowing the von Hippel-Lindau protein (pVHL) to bind, which targets HIFα for proteasomal degradation. During hypoxia, HIFα does not bind to pVHL and instead forms a transcriptional complex with HIFβ leading to increased expression of a broad range of hypoxia-regulated genes. Patients with Chuvash polycythemia (CP) are homozygous for a 598 C->T mutation in the VHL gene, resulting in a pVHL that causes ineffective degradation of HIFα. Clinically, CP patients suffer from premature mortality related to vascular thrombotic events - an increased risk that is not related to their elevated hematocrit, blood pressure, or known cardio or cerebrovascular risk factors. Lab abnormalities seen in CP include erythrocytosis and an elevated plasma glutathione level (Sergueeva et al, Haematologica. Feb 2008). Glutathione (GSH) plays an essential role in cellular antioxidant protection, and its levels are controlled by two mechanisms. GSH is oxidized to glutathione disulfide (GSSG) but is replenished by reduction of GSSG via glutathione reductase (GSR). GSH is also increased by de novo synthesis, which is regulated at several levels, including GSH feedback inhibition, and glutathione synthetase (GSS) and glutamate cysteine ligase (GCL) activity. To establish the molecular basis of elevated GSH, we examined expression of GSS, GSR, and GCL in the platelets of 11 CP patients and 8 Chuvash controls using qRT-PCR. Analysis revealed a 2-fold increased expression of GCL in CP. GSR and GSS were not statistically different. This data suggests that increased GCL activity might be the mechanism by which GSH is elevated in CP, but whether HIF directly regulates GCL or whether these differences reflect a more global process are presently unknown. Since the promoter of GCL contains an oxidative stressresponse element, transcriptional up regulation of GCL by increased oxidative stress secondary to HIF dysregulation might drive increased GSH synthesis. To examine this question, we measured GSSG in our samples. Although CP patients had elevated GSH (8.00 uM vs. 4.32 uM, p 0.002), the GSH/GSSG ratio (a widely used marker of redox state) showed no differences between CP and controls. Thus, overexpression of GCL and elevated GSH appear not to be compensatory responses to increased oxidative stress in CP. We also found increased GCL expression in VHL mutant mice. To determine if HIF1 might regulate this expression, we next measured GCL expression in HIF1 deficient embryos at embryonic day 9.5 and found decreased expression of GCL. In the homozygote knock-out embryos where HIF1 is absent, GCL expression is decreased, in contrast to CP patients, where HIF1 and GCL expression are both increased. Collectively, our data suggests that HIF1 dysregulates cellular redox homeostasis by upregulating GCL and increasing GSH synthesis in an oxidative-stress-independent manner. The significance of elevated GSH in CP and its possible relationship to increased thromboses remains to be defined. Reactive oxygen species mediate the vascular inflammation seen in the development of atherosclerotic disease, and GSH is an important intracellular scavenger that protects cells against oxidative damage. Yet CP patients have increased GSH as well as increased thrombosis, which is especially provoking since recently published evidence suggests that increased redox potential may be harmful to the cardiovascular system (Rajasekaran, et al Cell 2007). Perhaps the GSH elevation may contribute to the increased vascular disease that constitutes the major cause of mortality in this disorder of hypoxia sensing. The cellular antioxidant defense system is intimately linked to oxidative stress, hypoxia regulation, and vascular homeostasis. Our proposed future studies employing HIF2 knock out mice, model cell systems for HIF1 and HIF2, and the role of GSH in platelet function will be used to further explore the molecular mechanisms that regulate these complex pathways.

The ketogenic diet increases mitochondrial glutathione levels

The ketogenic diet (KD) is a high-fat, low carbohydrate diet that is used as a therapy for intractable epilepsy. However, the mechanism(s) by which the KD achieves neuroprotection and/or seizure control are not yet known. We sought to determine whether the KD improves mitochondrial redox status. Adolescent Sprague-Dawley rats (P28) were fed a KD or control diet for 3 weeks and ketosis was confirmed by plasma levels of beta-hydroxybutyrate (BHB). KD-fed rats showed a twofold increase in hippocampal mitochondrial GSH and GSH/GSSG ratios compared with control diet-fed rats. To determine whether elevated mitochondrial GSH was associated with increased de novo synthesis, the enzymatic activity of glutamate cysteine ligase (GCL) (the rate-limiting enzyme in GSH biosynthesis) and protein levels of the catalytic (GCLC) and modulatory (GCLM) subunits of GCL were analyzed. Increased GCL activity was observed in KD-fed rats, as well as up-regulated protein levels of GCL subunits. Reduced CoA (CoASH), an indicator of mitochondrial redox status, and lipoic acid, a thiol antioxidant, were also significantly increased in the hippocampus of KD-fed rats compared with controls. As GSH is a major mitochondrial antioxidant that protects mitochondrial DNA (mtDNA) against oxidative damage, we measured mitochondrial H2O2 production and H2O2-induced mtDNA damage. Isolated hippocampal mitochondria from KD-fed rats showed functional consequences consistent with the improvement of mitochondrial redox status i.e. decreased H2O2 production and mtDNA damage. Together, the results demonstrate that the KD up-regulates GSH biosynthesis, enhances mitochondrial antioxidant status, and protects mtDNA from oxidant-induced damage.

Modulation of antioxidant and detoxification responses mediated by lipoic acid in the fish Corydoras paleatus (Callychthyidae)

Lipoic acid (LA) has been reported as a potential therapeutic agent due its antioxidants proprieties. It was considered its effect in different organs (gills, brain, muscle and liver) of the fish Corydoras paleatus (Callychthyidae). LA (70 mg/kg of body mass) was added to a commercial fish diet, organisms being fed daily (1% body weight). Sixty animals (mean mass: 2.37 ± 0.09 g) were placed randomly in aquariums and received (+ LA) or not (− LA) lipoic acid enriched diet during four weeks. After, fish were killed and the brain, muscle, gills and liver were dissected. LA treatment reduced significantly ( p < 0.05) reactive oxygen species concentration in brain and increased ( p < 0.05) glutamate-cysteine ligase activity in brain and liver of the same experimental group. LA fed organisms showed higher ( p < 0.05) brain glutathione-S-transferase activity, indicating that LA improves the detoxification and antioxidant capacity face components that waste glutathione in phase II reactions. A conspicuous reduction of protein oxidation was observed in muscle and liver of + LA organisms, indicating that the treatment was also effective in reducing oxidative stress parameters.

Glutamate cysteine ligase (GCL) in the freshwater bivalve Unio tumidus: Impact of storage conditions and seasons on activity and identification of partial coding sequence of the catalytic subunit

Glutamate cysteine ligase (GCL; EC 6.3.2.2) is the first enzyme involved in the synthesis of glutathione. A HPLC method with fluorimetric detection was used to measure GCL activity in the gills and the digestive gland of the freshwater bivalve, Unio tumidus. Storage conditions were optimized in order to prevent decrease of GCL activity and consisted in freezing the cytosolic fraction in the presence of protease (1 mM phenylmethylsulfonic fluoric acid) and γ-glutamyltranspeptidase (1 mM L-serine borate mixture and 0.5 mM acivicin) inhibitors. Seasonal variations of activity in the digestive gland and to a lesser extent in the gills were found with activity increasing in spring compared to winter. No sex differences were revealed. The GCL coding sequence was identified using degenerated primers designed in the highly conserved regions of the catalytic subunit of GCL. The partial sequence identified encoded for 121 amino acids. The comparison of the identified partial coding sequence of U. tumidus with those available from vertebrates and invertebrates indicated that GCL sequence was highly conserved.

Optimization and application of glutamate cysteine ligase measurement in wildlife species

Glutamate cysteine ligase (GCL), synthesizing gamma-glutamylcysteine from glutamate and cysteine, is the rate-limiting enzyme in glutathione (GSH) biosynthesis. GCL activity measurement was optimized in tissues from deer mice, Sprague Dawley rats, and mallard ducks. Varying glutamic acid concentrations from 5 to 80 mM did not affect GCL activities markedly, whereas cysteine concentrations from 2.5 to 40 mM influenced GCL activities substantially. Optimal cysteine concentrations for deer mouse, Sprague Dawley rat, and mallard duck (respectively) were 30, 30, and 20 mM in liver, 10, 10, and 20 mM in kidney, 20, 20, and 30 mM in brain, and 30 mM in heart for all three species. Responses of mallard duck GCL activity to acid metalliferous water were evaluated. After subacute exposure, low doses increased GCL activity and GSH content in liver by 48.3% and 54.4%, respectively. High doses reduced GCL activities significantly in liver and kidney to 31.2% and 43.0% of the control, respectively.

Epigallocatechin-3-gallate Inhibits Growth of Activated Hepatic Stellate Cells by Enhancing the Capacity of Glutathione Synthesis

Activation of hepatic stellate cells (HSC), the key effectors in hepatic fibrogenesis, is characterized by enhanced cell proliferation and overproduction of extracellular matrix. Oxidative stress promotes HSC activation. Glutathione (GSH) is the most important intracellular antioxidant, whose synthesis is mainly regulated by glutamate-cysteine ligase (GCL). We reported previously that (-)-epigallocatechin-3-gallate (EGCG), the major and most active component in green tea extracts, inhibited HSC activation. The aim of this study is to elucidate the underlying mechanisms. We hypothesize that this inhibitory effect of EGCG might mainly result from its antioxidant capability by increasing de novo synthesis of GSH. In this report, we observe that EGCG enhances the levels of cytoplasmic and mitochondrial GSH and increases GCL activity by inducing gene expression of the catalytic subunit GCLc, leading to de novo synthesis of GSH. Real-time polymerase chain reaction and Western blotting analyses show that de novo synthesis of GSH is required for EGCG to regulate the expression of genes relevant to apoptosis and to cell proliferation. Additional experiments demonstrate that exogenous transforming growth factor (TGF)-beta1 suppresses GCLc gene expression and reduces the level of GSH in cultured HSC. Transient transfection assays and Western blotting analyses further display that EGCG interrupts TGF-beta signaling by reducing gene expression of TGF-beta receptors and Smad4, leading to increased expression of GCLc. These results support our hypothesis and collectively demonstrate that EGCG increases the level of cellular GSH in HSC by stimulating gene expression of GCLc, leading to the inhibition of cell proliferation of activated HSC in vitro.

Modulating GSH Synthesis Using Glutamate Cysteine Ligase Transgenic and Gene-Targeted Mice

Glutathione (GSH) is an important antioxidant and cofactor for glutathione S-transferase conjugation. GSH synthesis is catalyzed by glutamate cysteine ligase (GCL), composed of catalytic (GCLC) and modifier (GCLM) subunits. Transgenic mice that conditionally over express GCL subunits are protected from acetaminophen induced liver injury. Gclm null mice exhibit low GSH levels and enhanced sensitivity to acetaminophen. When Gclm expression and GCL activity are restored in Gclm conditional transgenic X Gclm null mice, they become resistant to APAP-induced liver damage. These animal models are a valuable resource for investigating the role of GSH synthesis in modulating oxidative damage and drug-induced hepatotoxicity.

γ-Tocopheryl quinone, not α-tocopheryl quinone, induces adaptive response through up-regulation of cellular glutathione and cysteine availability via activation of ATF4

α-Tocopheryl quinone (α-TQ) and γ-TQ are oxidized metabolites of the corresponding tocopherol (T) isoforms, which are vitamin E homologues. Unlike α-TQ, γ-TQ functions as an arylating agent that reacts with nucleophiles such as reduced sulphydryl groups and it has unique biological properties such as high toxicity. Increasing evidence indicates that reactive oxygen species and other physiologically existing oxidative stimuli upregulate the antioxidant system, thereby triggering the adaptive response. The present study used PC12 cells and immature primary cortical cells to examine the possible adaptive cytoprotective effects of γ-TQ against oxidative stress. Pre-treatment with γ-TQ at sub-lethal concentrations resulted in cytoprotective effects against oxidative stress. γ-TQ induced a significant increase in the cellular glutathione (GSH) levels while α-TQ did not. γ-TQ did not induce any considerable change in the activity of glutamate-cysteine ligase (GCL), the rate-limiting enzyme in GSH synthesis, whereas it increased the cellular GSH levels by facilitating the availability of cysteine through the induction of xCT, which is the core sub-unit of the xc− high-affinity cystine transporter system. An activating transcription factor 4 (ATF4)-small interfering RNA effectively attenuated the xCT mRNA level as well as the increase in cellular cysteine levels induced by γ-TQ, while the NF-E2-related factor (Nrf2)-small interfering RNA treatment did not. Collectively, these findings indicate that γ-TQ acts as a signal messenger to induce adaptive response through the upregulation of intracellular GSH synthesis via transcriptional activation of ATF4 in order to cope with the forthcoming oxidative insult.

Resveratrol induces glutathione synthesis by activation of Nrf2 and protects against cigarette smoke-mediated oxidative stress in human lung epithelial cells

Nuclear erythroid-related factor 2 (Nrf2), a redox-sensitive transcription factor, is involved in transcriptional regulation of many antioxidant genes, including glutamate-cysteine ligase (GCL). Cigarette smoke (CS) is known to cause oxidative stress and deplete glutathione (GSH) levels in alveolar epithelial cells. We hypothesized that resveratrol, a polyphenolic phytoalexin, has antioxidant signaling properties by inducing GSH biosynthesis via the activation of Nrf2 and protects lung epithelial cells against CS-mediated oxidative stress. Treatment of human primary small airway epithelial and human alveolar epithelial (A549) cells with CS extract (CSE) dose dependently decreased GSH levels and GCL activity, effects that were associated with enhanced production of reactive oxygen species. Resveratrol restored CSE-depleted GSH levels by upregulation of GCL via activation of Nrf2 and also quenched CSE-induced release of reactive oxygen species. Interestingly, CSE failed to induce nuclear translocation of Nrf2 in A549 and small airway epithelial cells. On the contrary, Nrf2 was localized in the cytosol of alveolar and airway epithelial cells due to CSE-mediated posttranslational modifications such as aldehyde/carbonyl adduct formation and nitration. On the other hand, resveratrol attenuated CSE-mediated Nrf2 modifications, thereby inducing its nuclear translocation associated with GCL gene transcription, as demonstrated by GCL-promoter reporter and Nrf2 small interfering RNA approaches. Thus resveratrol attenuates CSE-mediated GSH depletion by inducing GSH synthesis and protects epithelial cells by reversing CSE-induced posttranslational modifications of Nrf2. These data may have implications in dietary modulation of antioxidants in treatment of chronic obstructive pulmonary disease.

Impaired glutathione synthesis in schizophrenia: Convergent genetic and functional evidence

Schizophrenia is a complex multifactorial brain disorder with a genetic component. Convergent evidence has implicated oxidative stress and glutathione (GSH) deficits in the pathogenesis of this disease. The aim of the present study was to test whether schizophrenia is associated with a deficit of GSH synthesis. Cultured skin fibroblasts from schizophrenia patients and control subjects were challenged with oxidative stress, and parameters of the rate-limiting enzyme for the GSH synthesis, the glutamate cysteine ligase (GCL), were measured. Stressed cells of patients had a 26% (P = 0.002) decreased GCL activity as compared with controls. This reduction correlated with a 29% (P < 0.001) decreased protein expression of the catalytic GCL subunit (GCLC). Genetic analysis of a trinucleotide repeat (TNR) polymorphism in the GCLC gene showed a significant association with schizophrenia in two independent case-control studies. The most common TNR genotype 7/7 was more frequent in controls [odds ratio (OR) = 0.6, P = 0.003], whereas the rarest TNR genotype 8/8 was three times more frequent in patients (OR = 3.0, P = 0.007). Moreover, subjects with disease-associated genotypes had lower GCLC protein expression (P = 0.017), GCL activity (P = 0.037), and GSH contents (P = 0.004) than subjects with genotypes that were more frequent in controls. Taken together, the study provides genetic and functional evidence that an impaired capacity to synthesize GSH under conditions of oxidative stress is a vulnerability factor for schizophrenia.