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Terrance J Kavanagh - One of the best experts on this subject based on the ideXlab platform.
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functional significance of glutamate cysteine ligase modifier for erythrocyte survival in vitro and in vivo
Cell Death & Differentiation, 2013Co-Authors: M Foller, Terrance J Kavanagh, Isaac S Harris, Andrew J Elia, R John, Florian Lang, Tak W MakAbstract:Erythrocytes endure constant exposure to oxidative stress. The major oxidative stress scavenger in erythrocytes is glutathione. The rate-limiting enzyme for glutathione synthesis is glutamate–cysteine ligase, which consists of a catalytic subunit (GCLC) and a modifier subunit (GCLM). Here, we examined erythrocyte survival in GCLM-deficient (gclm−/−) mice. Erythrocytes from gclm−/− mice showed greatly reduced intracellular glutathione. Prolonged incubation resulted in complete lysis of gclm−/− erythrocytes, which could be reversed by exogenous delivery of the antioxidant Trolox. To test the importance of GCLM in vivo, mice were treated with phenylhydrazine (PHZ; 0.07 mg/g b.w.) to induce oxidative stress. Gclm−/− mice showed dramatically increased hemolysis compared with gclm+/+ controls. In addition, PHZ-treated gclm−/− mice displayed markedly larger accumulations of injured erythrocytes in the spleen than gclm+/+ mice within 24 h of treatment. Iron staining indicated precipitations of the erythrocyte-derived pigment hemosiderin in kidney tubules of gclm−/− mice and none in gclm+/+ controls. In fact, 24 h after treatment, kidney function began to diminish in gclm−/− mice as evident from increased serum creatinine and urea. Consequently, while all PHZ-treated gclm+/+ mice survived, 90% of PHZ-treated gclm−/− mice died within 5 days of treatment. In vitro, upon incubation in the absence or presence of additional oxidative stress, gclm−/− erythrocytes exposed significantly more phosphatidylserine, a cell death marker, than gclm+/+ erythrocytes, an effect at least partially due to increased cytosolic Ca2+ concentration. Under resting conditions, gclm−/− mice exhibited reticulocytosis, indicating that the enhanced erythrocyte death was offset by accelerated erythrocyte generation. GCLM is thus indispensable for erythrocyte survival, in vitro and in vivo, during oxidative stress.
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Rapid activation of glutamate cysteine ligase following oxidative stress
Journal of Biological Chemistry, 2010Co-Authors: Cecile M. Krejsa, Collin C White, Jeffrey A Ledbetter, Gary L. Schieven, Christopher C Franklin, Terrance J KavanaghAbstract:Glutamate cysteine ligase (GCL) catalyzes the rate-limiting step in the formation of the cellular antioxidant glutathione (GSH). The GCL holoenzyme consists of two separately coded proteins, a catalytic subunit (GCLC) and a modifier subunit (GCLM). Both GCLC and GLCM are controlled transcriptionally by a variety of cellular stimuli, including oxidative stress. This study addresses post-translational control of GCL activity, which increased rapidly in human lymphocytes following oxidative stress. Activation of GCL occurred within minutes of treatment and without any change in GCL protein levels and coincided with an increase in the proportion of GCLC in the holoenzyme form. Likewise, GCLM shifted from the monomeric form to holoenzyme and higher molecular weight species. Normal rat tissues also showed a distribution of monomeric and higher molecular weight forms. Neither GCL activation, nor the formation of holoenzyme, required a covalent intermolecular disulfide bridge between GCLC and GCLM. However, in immunoprecipitation studies, a neutralizing epitope associated with enzymatic activity was protected following cellular oxidative stress. Thus, the N-terminal portion of GCLC may undergo a change that stabilizes the GCL holoenzyme. Our results suggest that a dynamic equilibrium exists between low and high activity forms of GCL and is altered by transient oxidative stress. This provides a mechanism for the rapid post-translational activation of GCL and maintenance of cellular GSH homeostasis.
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distinct nrf1 2 independent mechanisms mediate as3 induced glutamate cysteine ligase subunit gene expression in murine hepatocytes
Free Radical Biology and Medicine, 2009Co-Authors: James A Thompson, Terrance J Kavanagh, Nelson Fausto, Charles L White, David P Cox, Jefferson Y Chan, Christopher C FranklinAbstract: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.
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glutamate cysteine ligase gcl transgenic and gene targeted mice for controlling glutathione synthesis
Current protocols in immunology, 2009Co-Authors: Isaac Mohar, Lisa A. Mcconnachie, Dianne Botta, Charles L White, Terrance J KavanaghAbstract:The tripeptide glutathione (GSH) has important antioxidant properties, scavenges free radicals, and serves as a cofactor for glutathione S-transferase conjugation of many xenobiotics. GSH is synthesized in two steps. The first and, often, rate-limiting step is the formation of γ-glutamylcysteine, which is catalyzed by the inducible heterodimeric enzyme glutamate cysteine ligase (GCL). The two subunits of GCL are the catalytic subunit (GCLC) and the modifier subunit (GCLM). In this unit, the generation and basic characterization methodologies of transgenic mouse models that have been developed to (1) conditionally over express both GCL subunits; (2) lack GCLM (Gclm null); and (3) create a hybrid between Gclm conditional over-expressing mice on a Gclm null genetic background are discussed. These models can be used to explore the fundamental role of GCLC and GCLM in GSH synthesis, as well as the toxicological role of GSH and its synthesis in xenobiotic metabolism and response to oxidative stress. Curr. Protoc. Toxicol. 39:6.16.1-6.16.20. © 2009 by John Wiley & Sons, Inc. Keywords: glutathione; glutamate cysteine ligase; oxidative stress; transgenic mouse; genetic model
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Modulating GSH Synthesis Using Glutamate Cysteine Ligase Transgenic and Gene-Targeted Mice
Drug Metabolism Reviews, 2008Co-Authors: Dianne Botta, Lisa A. Mcconnachie, Portia Vliet-gregg, Monica B. Mcgrath, Isaac Mohar, Collin C White, Terrance J KavanaghAbstract: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.
Henry Jay Forman - One of the best experts on this subject based on the ideXlab platform.
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silencing bach1 alters aging related changes in the expression of nrf2 regulated genes in primary human bronchial epithelial cells
Archives of Biochemistry and Biophysics, 2019Co-Authors: Hongqiao Zhang, Lulu Zhou, Kelvin J A Davies, Henry Jay FormanAbstract:Nrf2 is the master transcription factor regulating the basal and inducible expression of antioxidant genes. With aging, the basal Nrf2 activity is increased but oxidant/electrophile-enhanced activation of Nrf2 signaling is diminished, and these changes are accompanied by an increased expression of Bach1, a repressor of Nrf2 signaling. In this limited follow-up study, we explored how Bach1 may be involved in aging-related alteration in Nrf2 signaling in primary human bronchial epithelial (HBE) cells. Silencing Bach1 with siRNA increased the basal mRNA expression of Nrf2 regulated genes including glutamate cysteine ligase catalytic (GCLC) and modifier subunit (GCLM), NAD(P)H oxidoreductase 1(NQO-1) and heme oxygenase 1(HO-1), in HBE cells from both young (aged 21-29 years) and older (aged 61-69 years) donors. On the other hand, Bach1 silencing affected the induction of Nrf2-regulated genes differentially in young and older HBE cells. Bach1 silencing significantly enhanced sulforaphane-induced expression of HO-1 but had no effect on that of GCLC, GCLM, and NQO1 in young HBE cells. In contrast, Bach1 silencing enhanced sulforaphane-induced expression of GCLC, GCLM and HO-1 but had no effect on that of NQO-1 in older HBE cells. In conclusion, these results suggest that increased Bach1 contributes to aging-related loss of electrophile-enhanced Nrf2 signaling.
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down regulation of glutathione and glutamate cysteine ligase in the inflammatory response of macrophages
Free Radical Biology and Medicine, 2018Co-Authors: Hongqiao Zhang, Kelvin J A Davies, Sarah Jiuqi Zhang, Natalie Lyn, Abigail Florentino, Henry Jay FormanAbstract:Abstract Glutathione (GSH) plays critical roles in the inflammatory response by acting as the master substrate for antioxidant enzymes and an important anti-inflammatory agent. In the early phase of the inflammatory response of macrophages, GSH content is decreased due to the down regulation of the catalytic subunit of glutamate cysteine ligase (GCLC). In the current study we investigated the underlying mechanism for this phenomenon. In human THP1-differentiated macrophages, GCLC mRNA had a half-life of 4 h under basal conditions, and it was significantly reduced to less than 2 h upon exposure to lipopolysaccharide (LPS), suggesting an increased decay of GCLC mRNA in the inflammatory response. The half-life of GCLC protein was >10 h under basal conditions, and upon LPS exposure the degradation rate of GCLC protein was significantly increased. The pan-caspase inhibitor Z-VAD-FMK but not the proteasome inhibitor MG132, prevented the down regulation of GCLC protein caused by LPS. Both caspase inhibitor Z-LEVD-FMK and siRNA of caspase-5 abrogated LPS-induced degradation of GCLC protein. In addition, supplement with γ-GC, the GCLC product, efficiently restored GSH content and suppressed the induction of NF-κB activity by LPS. In conclusion, these data suggest that GCLC down-regulation in the inflammatory response of macrophages is mediated through both increased mRNA decay and caspase-5-mediated GCLC protein degradation, and γ-GC is an efficient agent to restore GSH and regulate the inflammatory response.
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association of gclm 588c t and GCLC 129t c promoter polymorphisms of genes coding the subunits of glutamate cysteine ligase with ischemic heart disease development in kazakhstan population
Disease Markers, 2017Co-Authors: Liliya Skvortsova, Henry Jay Forman, Anastasia Perfelyeva, Elmira Khussainova, Alma Mansharipova, Leyla DjansugurovaAbstract:Background. Glutamate cysteine ligase (GCL) is a rate-limiting enzyme in synthesis of glutathione. Evidence suggests that genetic variations in the promoter region of genes coding a catalytic subunit (GCLC -129T/C) and a modifier subunit (GCLM -588C/T) of GCL have a functional impact on their transcriptional activity and were associated with various disorders. Hence, we hypothesize whether these two polymorphic variants of GCLM and GCLC genes are associated with the risk of ischemic heart disease (IHD) development in the population of Kazakhstan. Methods. We evaluated 360 patients with IHD and 341 control subjects. Allele frequencies of studied promoters’ polymorphisms were detected by PCR-RFLP analysis. Multiple logistic regression analysis was applied to assess the risk for different genotypes obtained. Results. The presence of -588T allele in GCLM and -129T allele in GCLC gene genotypes was associated with an increased risk of IHD (GCLM -588T: , ; GCLC -129T: , ) for general ethnically mixed group. Analysis of each ethnical groups separately showed the higher risk tendency for Kazakhs as for GCLM -588T (; ) and as for GCLC -129T (, ). For Russians, statistically differences for two polymorphisms were not observed. Conclusion. The two promoter polymorphisms of GCLM (-588C/T) and GCLC (-128T/C) are associated with an increased risk of IHD in Kazakhstan population.
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competition of nuclear factor erythroid 2 factors related transcription factor isoforms nrf1 and nrf2 in antioxidant enzyme induction
Redox biology, 2013Co-Authors: Nikolai L Chepelev, Hongqiao Zhang, Henry Jay Forman, Kelvin J A Davies, Skye Mcbride, Andrew J Seal, Todd E Morgan, Caleb E Finch, William G WillmoreAbstract:Although the Nrf2 (nuclear factor-erythroid 2 p45 subunit-related factor 2) regulated expression of multiple antioxidant and cytoprotective genes through the electrophile responsive element (EpRE) is well established, interaction of Nrf2/EpRE with Nrf1, a closely-related transcription factor, is less well understood. Due to either proteolysis or alternative translation, Nrf1 has been found as proteins of varying size, p120, p95, and p65, which have been described as either activators of EpRE or competitive inhibitors of Nrf2. We investigated the effect of Nrf1 on EpRE-regulated gene expression using the catalytic and modifier subunits of glutamate cysteine ligase (GCLC and GCLM) as models and explored the potential role of Nrf1 in altering their expression in aging and upon chronic exposure to airborne nano-sized particulate matter (nPM). Nrf1 knockout resulted in the increased expression of GCLC and GCLM in human bronchial epithelial (HBE1) cells. Overexpression Nrf2 in combination with either p120 or p65 diminished or failed to further increase the GCLC- and GLCM-EpRE luciferase activity. All known forms of Nrf1 protein, remained unchanged in the lungs of mice with age or in response to nPM. Our study shows that Nrf1 could inhibit EpRE activity in vitro, whereas the precise role of Nrf1 in vivo requires further investigations. We conclude that Nrf1 may not be directly responsible for the loss of Nrf2-dependent inducibility of antioxidant and cytoprotective genes observed in aged animals.
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Structure, function, and post-translational regulation of the catalytic and modifier subunits of glutamate cysteine ligase.
Molecular aspects of medicine, 2008Co-Authors: Christopher C Franklin, Isaac Mohar, Collin C White, Donald S Backos, Henry Jay Forman, Terrance J KavanaghAbstract:Glutathione (GSH) is a tripeptide composed of glutamate, cysteine, and glycine. The first and rate-limiting step in GSH synthesis is catalyzed by glutamate cysteine ligase (GCL, previously known as gamma-glutamylcysteine synthetase). GCL is a heterodimeric protein composed of catalytic (GCLC) and modifier (GCLM) subunits that are expressed from different genes. GCLC catalyzes a unique gamma-carboxyl linkage from glutamate to cysteine and requires ATP and Mg(++) as cofactors in this reaction. GCLM increases the V(max) and K(cat) of GCLC, decreases the K(m) for glutamate and ATP, and increases the K(i) for GSH-mediated feedback inhibition of GCL. While post-translational modifications of GCLC (e.g. phosphorylation, myristoylation, caspase-mediated cleavage) have modest effects on GCL activity, oxidative stress dramatically affects GCL holoenzyme formation and activity. Pyridine nucleotides can also modulate GCL activity in some species. Variability in GCL expression is associated with several disease phenotypes and transgenic mouse and rat models promise to be highly useful for investigating the relationships between GCL activity, GSH synthesis, and disease in humans.
Ulrike Luderer - One of the best experts on this subject based on the ideXlab platform.
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overexpression of glutamate cysteine ligase protects human cov434 granulosa tumour cells against oxidative and γ radiation induced cell death
Mutagenesis, 2009Co-Authors: Mabel M Corteswanstreet, Erich Giedzinski, Charles L Limoli, Ulrike LudererAbstract:Ionizing radiation is toxic to ovarian follicles and can cause infertility. Generation of reactive oxygen species (ROS) has been implicated in the toxicity of ionizing radiation in several cell types. We have shown that depletion of the antioxidant glutathione (GSH) sensitizes follicles and granulosa cells to toxicant-induced apoptosis and that supplementation of GSH is protective. The rate-limiting reaction in GSH biosynthesis is catalysed by glutamate-cysteine ligase (GCL), which consists of a catalytic subunit (GCLC) and a regulatory subunit (GCLM). We hypothesized that overexpression of GCLC or Gclm to increase GSH synthesis would protect granulosa cells against oxidant- and radiation-induced cell death. The COV434 line of human granulosa tumour cells was stably transfected with vectors designed for the constitutive expression of GCLC, Gclm, both GCLC and Gclm or empty vector. GCL protein and enzymatic activity and total GSH levels were significantly increased in the GCL subunit-transfected cells. GCL-transfected cells were resistant to cell killing by treatment with hydrogen peroxide compared to control cells. Cell viability declined less in all the GCL subunit-transfected cell lines 1-8 h after 0.5 mM hydrogen peroxide treatment than in control cells. We next examined the effects of GCL overexpression on responses to ionizing radiation. ROS were measured using a redox-sensitive fluorogenic dye in cells irradiated with 0, 1 or 5 Gy of gamma-rays. There was a dose-dependent increase in ROS within 30 min in all cell lines, an effect that was significantly attenuated in Gcl-transfected cells. Apoptosis, assessed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelling and activated caspase-3 immunoblotting, was significantly decreased in irradiated GCLC-transfected cells compared to irradiated control cells. Suppression of GSH synthesis in GCLC-transfected cells reversed resistance to radiation. These findings show that overexpression of GCL in granulosa cells can augment GSH synthesis and ameliorate various sequelae associated with exposure to oxidative stress and irradiation.
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Overexpression of glutamate-cysteine ligase protects human COV434 granulosa tumour cells against oxidative and gamma-radiation-induced cell death
2009Co-Authors: Mabel M. Cortes-wanstreet, Erich Giedzinski, L. Limoli, Ulrike LudererAbstract:Ionizing radiation is toxic to ovarian follicles and can cause infertility. Generation of reactive oxygen species (ROS) has been implicated in the toxicity of ionizing radiation in several cell types. We have shown that depletion of the antioxidant glutathione (GSH) sensitizes follicles and granulosa cells to toxicant-induced apoptosis and that supplementation of GSH is protective. The rate-limiting reaction in GSH biosynthesis is catalysed by glutamate– cysteine ligase (GCL), which consists of a catalytic subunit (GCLC) and a regulatory subunit (GCLM). We hypothe-sized that overexpression of GCLC or Gclm to increase GSH synthesis would protect granulosa cells against oxidant-and radiation-induced cell death. The COV434 line of human granulosa tumour cells was stably transfected with vectors designed for the constitutive expression of GCLC
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gonadotropin regulation of glutamate cysteine ligase catalytic and modifier subunit expression in rat ovary is subunit and follicle stage specific
American Journal of Physiology-endocrinology and Metabolism, 2005Co-Authors: Miyun Tsaiturton, Ulrike LudererAbstract:We have observed that levels of the antioxidant glutathione (GSH) and protein levels of the catalytic and modifier subunits of the rate-limiting enzyme in GSH synthesis, GCLC and GCLm, increase in ...
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Localization of glutamate cysteine ligase subunit mRNA within the rat ovary and relationship to follicular apoptosis
Molecular Reproduction and Development, 2003Co-Authors: Ulrike Luderer, Dolores Diaz, Elaine M Faustman, Terrance J KavanaghAbstract:Ovarian levels of the antioxidant tripeptide glutathione (GSH) increase following gonadotropin administration, suggesting that GSH synthesis in the ovary may be associated with follicular growth. In situ hybridization with 35S-labeled riboprobes was used to localize ovarian mRNA expression of the catalytic and modulatory subunits of glutamate cysteine ligase (GCLC and Gclm), the rate-limiting enzyme in GSH synthesis, during each stage of the rat estrous cycle. Gclm was highly expressed in the granulosa cells and oocytes of healthy, growing follicles, not in atretic follicles. GCLC was also highly expressed in follicles; however, unlike Gclm, GCLC was also expressed in corpora lutea and interstitial cells. In a subsequent experiment, the hypothesis that GSH synthesis occurs in healthy, but not in apoptotic, follicles was tested. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) was used to detect apoptotic cells in the ovaries, and in situ hybridization for Gclm and GCLC was performed in adjacent sections of the same ovaries. TUNEL staining was found to be significantly associated with absence of Gclm hybridization in granulosa cells and oocytes and with lack of strong GCLC hybridization in granulosa cells. These results suggest that follicular apoptosis may be associated with down-regulation of Gclm and GCLC transcription in granulosa cells and oocytes. Mol. Reprod. Dev. 65: 254–261, 2003. © 2003 Wiley-Liss, Inc.
Timothy P Dalton - One of the best experts on this subject based on the ideXlab platform.
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Early onset senescence occurs when fibroblasts lack the glutamate-cysteine ligase modifier subunit.
Free Radical Biology and Medicine, 2009Co-Authors: Ying Chen, Vasilis Vasiliou, Howard G Shertzer, Elisabet Johansson, Timothy P DaltonAbstract:Abstract Cellular senescence is the irreversible entry of cells into growth arrest. Senescence of primary cells in culture has long been used as an in vitro model for aging. Glutamate–cysteine ligase (GCL) controls the synthetic rate of the important cellular antioxidant glutathione (GSH). The catalytic subunit of GCL, GCLC, is catalytically active and essential for life. By contrast the modifier subunit of GCL, GCLM, is dispensable in mice. Although it is recognized that GCLM increases the rate of GSH synthesis, its physiological role is unclear. Herein, we show that loss of Gclm leads to premature senescence of primary murine fibroblasts as characterized by: (a) diminished growth rate, (b) cell morphology consistent with senescence, (c) increases in senescence-associated β-galactosidase activity, and (d) cell cycle arrest at the G 1 /S and G 2 /M boundaries. These changes are accompanied by increased intracellular ROS, accumulation of DNA damage, and induction of p53 and p21 proteins. We also found that N- acetylcysteine increases intracellular GSH and prevents premature senescence in Gclm −/− cells. These results suggest that the control of GCLM, which in turn controls aspects of the cellular redox environment via GSH, is important in determining the replicative capacity of the cell.
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Interaction between the catalytic and modifier subunits of glutamate-cysteine ligase
Biochemical Pharmacology, 2007Co-Authors: Yi Yang, Scott N. Schneider, Howard G Shertzer, Elisabet Johansson, Ying Chen, Timothy P DaltonAbstract:Abstract Glutamate-cysteine ligase (GCL) is the rate-limiting enzyme in the glutathione (GSH) biosynthesis pathway. This enzyme is a heterodimer, comprising a catalytic subunit (GCLC) and a regulatory subunit (GCLM). Although GCLC alone can catalyze the formation of l -γ-glutamyl- l -cysteine, its binding with GCLM enhances the enzyme activity by lowering the K m for glutamate and ATP, and increasing the K i for GSH inhibition. To characterize the enzyme structure–function relationship, we investigated the heterodimer formation between GCLC and GCLM, in vivo using the yeast two-hybrid system, and in vitro using affinity chromatography. A strong and specific interaction between GCLC and GCLM was observed in both systems. Deletion analysis indicated that most regions, except a portion of the C-terminal region of GCLC and a portion of the N-terminal region of GCLM, are required for the interaction to occur. Point mutations of selected amino acids were also tested for the binding activity. The GCLC Cys248Ala/Cys249Ala and Pro158Leu mutations enzyme showed the same strength of binding to GCLM as did wild-type GCLC, yet the catalytic activity was dramatically decreased. The results suggest that the heterodimer formation may not be dependent on primary amino-acid sequence but, instead, involves a complex formation of the tertiary structure of both proteins.
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Glutamate Cysteine Ligase Catalysis DEPENDENCE ON ATP AND MODIFIER SUBUNIT FOR REGULATION OF TISSUE GLUTATHIONE LEVELS
Journal of Biological Chemistry, 2005Co-Authors: Ying Chen, Scott N. Schneider, Howard G Shertzer, Timothy P DaltonAbstract:Abstract Glutamate cysteine ligase (GCL), which synthesizes γ-glutamyl-cysteine (γ-GC), is the rate-limiting enzyme in GSH biosynthesis. γ-GC may be produced by the catalytic subunit GCLC or by the holoenzyme (GCLholo), which comprises GCLC and the modifier subunit GCLM. The Gclm(–/–) knock-out mouse shows tissue levels of GSH that are between 9 and 40% of the Gclm(+/+) wild-type mouse. In the present study, we used recombinant GCLC and GCLM and Gclm(–/–) mice to examine the role of GCLM on γ-GC synthesis by GCLholo. GCLM decreased the Km for ATP by ∼6-fold and, similar to other species, decreased the Km for glutamate and increased the Ki for feedback inhibition by GSH. Furthermore, GCLM increased by 4.4-fold the Kcat for γ-GC synthesis; this difference in catalytic efficiency of GCLholo versus GCLC allowed us to derive a mathematical relationship for γ-GC production and to determine the relative levels of GCLholo and GCLC; in homogenates of brain, liver, and lung, the ratio of GCLC to GCLholo was 7.0, 2.0, and 3.5, respectively. In kidney, however, the relationship between GCLC and GCLholo was complicated. Kidney contains GCLholo, free GCLC, and free GCLM, and free GCLC in kidney cannot interact with GCLM. Taken together, we conclude that, in most tissues, GCLM is limiting, suggesting that an increase in GCLM alone would increase γ-GC synthesis. On the other hand, our results from kidney suggest that γ-GC synthesis may be controlled post-translationally.
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initial characterization of the glutamate cysteine ligase modifier subunit gclm knockout mouse novel model system for a severely compromised oxidative stress response
Journal of Biological Chemistry, 2002Co-Authors: Yi Yang, Howard G Shertzer, Ying Chen, Matthew Z Dieter, Daniel W Nebert, Timothy P DaltonAbstract:Glutamate-cysteine ligase (GCL) is the rate-limiting enzyme in the GSH biosynthesis pathway. In higher eukaryotes, this enzyme is a heterodimer comprising a catalytic subunit (GCLC) and a modifier subunit (GCLM), which change the catalytic characteristics of the holoenzyme. To define the cellular function of GCLM, we disrupted the mouse Gclm gene to create a null allele. Gclm(-/-) mice are viable and fertile and have no overt phenotype. In liver, lung, pancreas, erythrocytes, and plasma, however, GSH levels in Gclm(-/-) mice were 9-16% of that in Gclm(+/+) littermates. Cysteine levels in Gclm(-/-) mice were 9, 35, and 40% of that in Gclm(+/+) mice in kidney, pancreas, and plasma, respectively, but remained unchanged in the liver and erythrocytes. Comparing the hepatic GCL holoenzyme with GCLC in the genetic absence of GCLM, we found the latter had an approximately 2-fold increase in K(m) for glutamate and a dramatically enhanced sensitivity to GSH inhibition. The major decrease in GSH, combined with diminished GCL activity, rendered Gclm(-/-) fetal fibroblasts strikingly more sensitive to chemical oxidants such as H(2)O(2). We conclude that the Gclm(-/-) mouse represents a model of chronic GSH depletion that will be very useful in evaluating the role of the GCLM subunit and GSH in numerous pathophysiological conditions as well as in environmental toxicity associated with oxidant insult.
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glutamate cysteine ligase modifier subunit mouse gclm gene structure and regulation by agents that cause oxidative stress
Biochemical Pharmacology, 2002Co-Authors: Willy A Solis, Timothy P Dalton, Howard G Shertzer, Matthew Z Dieter, Sarah L Freshwater, Judy M Harrer, Daniel W NebertAbstract:Abstract Glutamate–cysteine ligase is a heterodimer comprising a modifier (GCLM) and a catalytic (GCLC) subunit. In mouse Hepa-1c1c7 hepatoma cell cultures, we found that tert-butylhydroquinone (tBHQ; 50 μM) induces the GCLM and GCLC mRNAs ∼10- and ∼2-fold, respectively, and that these increases primarily reflect de novo transcription. We determined that the mouse Gclm gene has seven exons, spanning 22.3 kb; all exons, intron–exon junctions, and 4.7 kb of 5′-flanking region were sequenced. By RNase protection analysis, we identified two major and several minor transcription start-site clusters over a 300-bp region. The Gclm 5′-flanking region is GC-rich and lacks a canonical TATA box. Transient and stable transfection studies, using luciferase reporter constructs containing incremental Gclm 5′-flanking deletions (4.7–0.5 kb), showed high basal activity but only modest (∼2-fold) inducibility by tBHQ. The only candidate motif for oxidative stress regulation (in the 4.7-kb region we sequenced) is a putative inverted electrophile response element (EPRE) 9 bp upstream from the 5′-most transcription start-site. Site-directed mutagenesis of this −9 EPRE demonstrated minimal (30–40%) decreases in tBHQ induction and no effect on basal activity—suggesting that this EPRE might be necessary but not sufficient. The nuclear erythroid factor-2 (NEF2)-related factor-2 (NRF2) is known to transactivate via EPRE motifs. In the presence of co-transfected NRF cDNA expression vector, however, no increase in Gclm promoter activity was observed. Thus, the endogenous Gclm gene shows robust transcriptional activation by tBHQ in the intact Hepa-1 cell, but reporter constructs containing up to 4.7 kb of promoter (having only the one EPRE at −9) demonstrate a disappointing response, indicating that the major tBHQ-responsive regulatory element of the mouse Gclm gene must exist either further 5′- or 3′-ward of the 4.7-kb region studied.
Christopher C Franklin - One of the best experts on this subject based on the ideXlab platform.
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Glycation of Glutamate Cysteine Ligase by 2-Deoxy- d -Ribose and its Potential Impact on Chemoresistance in Glioblastoma
Neurochemical Research, 2013Co-Authors: Donald S Backos, Kristofer S. Fritz, Debbie G. Mcarthur, Andrew M. Donson, Nicholas K. Foreman, Jadwiga K Kepa, Christopher C Franklin, Philip ReiganAbstract:The antioxidant glutathione (GSH) plays a critical role in maintaining intracellular redox homeostasis but in tumors the GSH biosynthetic pathway is often dysregulated, contributing to tumor resistance to radiation and chemotherapy. Glutamate-cysteine ligase (GCL) catalyzes the first and rate-limiting reaction in GSH synthesis, and enzyme function is controlled by GSH feedback inhibition or by transcriptional upregulation of the catalytic (GCLC) and modifier (GCLM) subunits. However, it has recently been reported that the activity of GCLC and the formation of GCL can be modified by reactive aldehyde products derived from lipid peroxidation. Due to the susceptibility of GCLC to posttranslational modifications by reactive aldehydes, we examined the potential for 2-deoxy-d-ribose (2dDR) to glycate GCLC and regulate enzyme activity and GCL formation. 2dDR was found to directly modify both GCLC and GCLM in vitro, resulting in a significant inhibition of GCLC and GCL enzyme activity without altering substrate affinity or feedback inhibition. 2dDR-mediated glycation also inhibited GCL subunit heterodimerization and formation of the GCL holoenzyme complex while not causing dissociation of pre-formed holoenzyme. This PTM could be of particular importance in glioblastoma (GBM) where intratumoral necrosis provides an abundance of thymidine, which can be metabolized by thymidine phosphorylase (TP) to form 2dDR. TP is expressed at high levels in human GBM tumors and shRNA knockdown of TP in U87 GBM cells results in a significant increase in cellular GCL enzymatic activity.
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Posttranslational modification and regulation of glutamate-cysteine ligase by the α,β-unsaturated aldehyde 4-hydroxy-2-nonenal
Free Radical Biology and Medicine, 2010Co-Authors: Donald S Backos, Kristofer S. Fritz, James R Roede, Christopher C FranklinAbstract:Abstract 4-Hydroxy-2-nonenal (4-HNE) is a lipid peroxidation product formed during oxidative stress that can alter protein function via adduction of nucleophilic amino acid residues. 4-HNE detoxification occurs mainly via glutathione (GSH) conjugation and transporter-mediated efflux. This results in a net loss of cellular GSH, and restoration of GSH homeostasis requires de novo GSH biosynthesis. The rate-limiting step in GSH biosynthesis is catalyzed by glutamate–cysteine ligase (GCL), a heterodimeric holoenzyme composed of a catalytic (GCLC) and a modulatory (GCLM) subunit. The relative levels of the GCL subunits are a major determinant of cellular GSH biosynthetic capacity and 4-HNE induces the expression of both GCL subunits. In this study, we demonstrate that 4-HNE can alter GCL holoenzyme formation and activity via direct posttranslational modification of the GCL subunits in vitro. 4-HNE directly modified Cys553 of GCLC and Cys35 of GCLM in vitro, which significantly increased monomeric GCLC enzymatic activity, but reduced GCL holoenzyme activity and formation of the GCL holoenzyme complex. In silico molecular modeling studies also indicate these residues are likely to be functionally relevant. Within a cellular context, this novel posttranslational regulation of GCL activity could significantly affect cellular GSH homeostasis and GSH-dependent detoxification during periods of oxidative stress.
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Rapid activation of glutamate cysteine ligase following oxidative stress
Journal of Biological Chemistry, 2010Co-Authors: Cecile M. Krejsa, Collin C White, Jeffrey A Ledbetter, Gary L. Schieven, Christopher C Franklin, Terrance J KavanaghAbstract:Glutamate cysteine ligase (GCL) catalyzes the rate-limiting step in the formation of the cellular antioxidant glutathione (GSH). The GCL holoenzyme consists of two separately coded proteins, a catalytic subunit (GCLC) and a modifier subunit (GCLM). Both GCLC and GLCM are controlled transcriptionally by a variety of cellular stimuli, including oxidative stress. This study addresses post-translational control of GCL activity, which increased rapidly in human lymphocytes following oxidative stress. Activation of GCL occurred within minutes of treatment and without any change in GCL protein levels and coincided with an increase in the proportion of GCLC in the holoenzyme form. Likewise, GCLM shifted from the monomeric form to holoenzyme and higher molecular weight species. Normal rat tissues also showed a distribution of monomeric and higher molecular weight forms. Neither GCL activation, nor the formation of holoenzyme, required a covalent intermolecular disulfide bridge between GCLC and GCLM. However, in immunoprecipitation studies, a neutralizing epitope associated with enzymatic activity was protected following cellular oxidative stress. Thus, the N-terminal portion of GCLC may undergo a change that stabilizes the GCL holoenzyme. Our results suggest that a dynamic equilibrium exists between low and high activity forms of GCL and is altered by transient oxidative stress. This provides a mechanism for the rapid post-translational activation of GCL and maintenance of cellular GSH homeostasis.
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distinct nrf1 2 independent mechanisms mediate as3 induced glutamate cysteine ligase subunit gene expression in murine hepatocytes
Free Radical Biology and Medicine, 2009Co-Authors: James A Thompson, Terrance J Kavanagh, Nelson Fausto, Charles L White, David P Cox, Jefferson Y Chan, Christopher C FranklinAbstract: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.
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Structure, function, and post-translational regulation of the catalytic and modifier subunits of glutamate cysteine ligase.
Molecular aspects of medicine, 2008Co-Authors: Christopher C Franklin, Isaac Mohar, Collin C White, Donald S Backos, Henry Jay Forman, Terrance J KavanaghAbstract:Glutathione (GSH) is a tripeptide composed of glutamate, cysteine, and glycine. The first and rate-limiting step in GSH synthesis is catalyzed by glutamate cysteine ligase (GCL, previously known as gamma-glutamylcysteine synthetase). GCL is a heterodimeric protein composed of catalytic (GCLC) and modifier (GCLM) subunits that are expressed from different genes. GCLC catalyzes a unique gamma-carboxyl linkage from glutamate to cysteine and requires ATP and Mg(++) as cofactors in this reaction. GCLM increases the V(max) and K(cat) of GCLC, decreases the K(m) for glutamate and ATP, and increases the K(i) for GSH-mediated feedback inhibition of GCL. While post-translational modifications of GCLC (e.g. phosphorylation, myristoylation, caspase-mediated cleavage) have modest effects on GCL activity, oxidative stress dramatically affects GCL holoenzyme formation and activity. Pyridine nucleotides can also modulate GCL activity in some species. Variability in GCL expression is associated with several disease phenotypes and transgenic mouse and rat models promise to be highly useful for investigating the relationships between GCL activity, GSH synthesis, and disease in humans.