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Terrance J Kavanagh - One of the best experts on this subject based on the ideXlab platform.

  • Glutamate Cysteine Ligase modifier subunit gclm null mice have increased ovarian oxidative stress and accelerated age related ovarian failure
    Endocrinology, 2015
    Co-Authors: Jinhwan Lim, Isaac Mohar, Terrance J Kavanagh, Brooke N Nakamura, Ulrike Luderer
    Abstract:

    Glutathione (GSH) is the one of the most abundant intracellular antioxidants. Mice lacking the modifier subunit of Glutamate Cysteine Ligase (Gclm), the rate-limiting enzyme in GSH synthesis, have decreased GSH. Our prior work showed that GSH plays antiapoptotic roles in ovarian follicles. We hypothesized that Gclm(-/-) mice have accelerated ovarian aging due to ovarian oxidative stress. We found significantly decreased ovarian GSH concentrations and oxidized GSH/oxidized glutathione redox potential in Gclm(-/-) vs Gclm(+/+) ovaries. Prepubertal Gclm(-/-) and Gclm(+/+) mice had similar numbers of ovarian follicles, and as expected, the total number of ovarian follicles declined with age in both genotypes. However, the rate of decline in follicles was significantly more rapid in Gclm(-/-) mice, and this was driven by accelerated declines in primordial follicles, which constitute the ovarian reserve. We found significantly increased 4-hydroxynonenal immunostaining (oxidative lipid damage marker) and significantly increased nitrotyrosine immunostaining (oxidative protein damage marker) in prepubertal and adult Gclm(-/-) ovaries compared with controls. The percentage of small ovarian follicles with increased granulosa cell proliferation was significantly higher in prepubertal and 2-month-old Gclm(-/-) vs Gclm(+/+) ovaries, indicating accelerated recruitment of primordial follicles into the growing pool. The percentages of growing follicles with apoptotic granulosa cells were increased in young adult ovaries. Our results demonstrate increased ovarian oxidative stress and oxidative damage in young Gclm(-/-) mice, associated with an accelerated decline in ovarian follicles that appears to be mediated by increased recruitment of follicles into the growing pool, followed by apoptosis at later stages of follicular development.

  • functional significance of Glutamate Cysteine Ligase modifier for erythrocyte survival in vitro and in vivo
    Cell Death & Differentiation, 2013
    Co-Authors: M Foller, Terrance J Kavanagh, Isaac S Harris, Andrew J Elia, R John, Florian Lang, Tak W Mak
    Abstract:

    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.

  • transforming growth factor β suppresses Glutamate Cysteine Ligase gene expression and induces oxidative stress in a lung fibrosis model
    Free Radical Biology and Medicine, 2012
    Co-Authors: Ruiming Liu, Terrance J Kavanagh, Dale A Dickinson, Praveen K Vayalil, Carol A Ballinger, Wen Tan Huang, Suqing Wang, Qiana L Matthews, Edward M Postlethwait
    Abstract:

    The concentration of glutathione (GSH), the most abundant intracellular free thiol and an important antioxidant, is decreased in the lung in both fibrotic diseases and experimental fibrosis models. The underlying mechanisms and biological significance of GSH depletion, however, remain unclear. Transforming growth factor β (TGF-β) is the most potent and ubiquitous profibrogenic cytokine and its expression is increased in almost all fibrotic diseases. In this study, we show that increasing TGF-β1 expression in mouse lung to a level comparable to those found in lung fibrotic diseases by intranasal instillation of AdTGF-β1(223/225), an adenovirus expressing constitutively active TGF-β1, suppressed the expression of both catalytic and modifier subunits of Glutamate-Cysteine Ligase (GCL), the rate-limiting enzyme in de novo GSH synthesis, decreased GSH concentration, and increased protein and lipid peroxidation in mouse lung. Furthermore, we show that increasing TGF-β1 expression activated JNK and induced activating transcription factor 3, a transcriptional repressor involved in the regulation of the catalytic subunit of GCL, in mouse lung. Control virus (AdDL70-3) had no significant effect on any of these parameters, compared to saline-treated control. Concurrent with GSH depletion, TGF-β1 induced lung epithelial apoptosis and robust pulmonary fibrosis. Importantly, lung GSH levels returned to normal, whereas fibrosis persisted at least 21 days after TGF-β1 instillation. Together, the data suggest that increased TGF-β1 expression may contribute to the GSH depletion observed in pulmonary fibrosis diseases and that GSH depletion may be an early event in, rather than a consequence of, fibrosis development.

  • increased sensitivity to testicular toxicity of transplacental benzo a pyrene exposure in male Glutamate Cysteine Ligase modifier subunit knockout gclm mice
    Toxicological Sciences, 2012
    Co-Authors: Brooke N Nakamura, Lisa A. Mcconnachie, Isaac Mohar, Yvonne D Hoang, Gregory W Lawson, Mabel M Cortes, Laura Ortiz, Reshma Patel, Bogdan A Rau, Terrance J Kavanagh
    Abstract:

    Polycyclic aromatic hydrocarbons (PAHs), like benzo[a]pyrene (BaP), are ubiquitous environmental pollutants formed by the incomplete combustion of organic materials. The tripeptide glutathione (GSH) is a major antioxidant and is important in detoxification of PAH metabolites. Mice null for the modifier subunit of Glutamate Cysteine Ligase (Gclm), the rate-limiting enzyme in GSH synthesis, have decreased GSH concentrations. We investigated the effects of Gclm deletion alone on male fertility and spermatogenesis and its effect on the sensitivity of male embryos to the transplacental testicular toxicity of BaP. Gclm-/- males had dramatically decreased testicular and epididymal GCL enzymatic activity and total GSH concentrations compared with Gclm+/+ littermates. Ratios of reduced to oxidized GSH were significantly increased in Gclm-/- testes. GSH reductase enzymatic activity was increased in Gclm-/- epididymides. We observed no changes in fertility, testicular weights, testicular sperm head counts, or testicular histology and subtle changes in cauda epididymal sperm counts, motility, and morphology in Gclm-/- compared with Gclm+/+ males. Prenatal exposure to BaP from gestational day 7 to 16 was dose dependently associated with significantly decreased testicular and epididymal weights, testicular and epididymal sperm counts, and with vacuolated seminiferous tubules at 10 weeks of age. Gclm-/- males exposed prenatally to BaP had greater decreases in testicular weights, testicular sperm head counts, epididymal sperm counts, and epididymal sperm motility than Gclm+/+ littermates. These results show no effects of Gclm deletion alone on male fertility and testicular spermatogenesis and subtle epididymal effects but support increased sensitivity of Gclm-/- males to the transplacental testicular toxicity of BaP.

  • lack of maternal Glutamate Cysteine Ligase modifier subunit gclm decreases oocyte glutathione concentrations and disrupts preimplantation development in mice
    Endocrinology, 2011
    Co-Authors: Brooke N Nakamura, Lisa A. Mcconnachie, Terrance J Kavanagh, Thomas J Fielder, Yvonne D Hoang, Jinhwan Lim, Ulrike Luderer
    Abstract:

    Glutathione (GSH) is the most abundant intracellular thiol and an important regulator of cellular redox status. Mice that lack the modifier subunit of Glutamate Cysteine Ligase (Gclm), the rate-limiting enzyme in GSH synthesis, have decreased GSH synthesis. Nicotinamide nucleotide transhydrogenase, an inner mitochondrial membrane protein, catalyzes the interconversion of reduced nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide phosphate; reduced nicotinamide adenine dinucleotide phosphate is required for reduction of GSH disulfide. Previous work supports roles for GSH in preimplantation development. We hypothesized that Gclm−/− mice have increased preimplantation embryonic mortality and that this effect is enhanced by absence of a functioning Nnt gene. Gclm−/− females produced significantly fewer pups per litter than Gclm+/+ littermates. Numbers of oocytes ovulated in a natural estrous cycle or upon superovulation did not differ by genotype. Fewer uterine implantation sites were observed in the Gclm−/− females. Prepubertal Gclm−/− and Gclm+/+ females were superovulated, then mated overnight with a Gclm+/+ male. At 0.5 d postcoitum, Gclm−/− females had significantly lower percentages of zygotes with two pronuclei and higher percentages of zygotes with one pronucleus than Gclm+/+ or Gclm+/− females. At 3.5 d postcoitum, a significantly lower percentage of blastocyst stage embryos was recovered from uteri of Gclm−/− females than Gclm+/+ females. Embryonic development to the blastocyst stage, but not the two-cell stage, was significantly decreased after in vitro fertilization of oocytes from Gclm−/− females compared with Gclm+/+ females. The Nnt mutation did not enhance the effects of Gclm genotype on female fertility. These results demonstrate critical roles for maternal GSH in supporting normal preimplantation development.

Henry Jay Forman - One of the best experts on this subject based on the ideXlab platform.

  • down regulation of glutathione and Glutamate Cysteine Ligase in the inflammatory response of macrophages
    Free Radical Biology and Medicine, 2018
    Co-Authors: Hongqiao Zhang, Sarah Jiuqi Zhang, Natalie Lyn, Abigail Florentino, Kelvin J A Davies, Henry Jay Forman
    Abstract:

    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.

  • Resveratrol and 4-hydroxynonenal act in concert to increase Glutamate Cysteine Ligase expression and glutathione in human bronchial epithelial cells.
    Archives of Biochemistry and Biophysics, 2008
    Co-Authors: Hongqiao Zhang, Alessandra Rinna, Albert Shih, Henry Jay Forman
    Abstract:

    Resveratrol has been shown to protect against oxidative stress through modulating antioxidant capacity. In this study, we investigated resveratrol-mediated induction of glutathione (GSH) and Glutamate Cysteine Ligase (GCL), and the combined effect of resveratrol and 4-hydroxynonenal (HNE) on GSH synthesis in cultured HBE1 human bronchial epithelial cells. Resveratrol increased GSH and the mRNA contents of both the catalytic (GCLC) and modulatory subunit (GCLM) of GCL. Combined HNE and resveratrol treatment increased GSH content and GCL mRNAs to a greater extent than either compound did alone. Compared to individual agent, combining exposure to HNE and resveratrol also showed more protection against cell death caused by oxidative stress. These effects of combined exposure were additive rather than synergistic. In addition, Nrf2 silencing significantly decreased the combined effect of HNE and resveratrol on GCL induction. Our data suggest that resveratrol increases GSH and GCL gene expression and that there is an additive effect on GSH synthesis between resveratrol and HNE. The results also reveal that Nrf2-EpRE signaling was involved in the combined effects.

  • Structure, function, and post-translational regulation of the catalytic and modifier subunits of Glutamate Cysteine Ligase.
    Molecular aspects of medicine, 2008
    Co-Authors: Christopher C Franklin, Isaac Mohar, Collin C White, Donald S Backos, Henry Jay Forman, Terrance J Kavanagh
    Abstract:

    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.

  • Hyperthermic stress-induced increase in the expression of Glutamate-Cysteine Ligase and glutathione levels in the symbiotic sea anemone Aiptasia pallida
    Comparative Biochemistry and Physiology B, 2008
    Co-Authors: Shinichi Sunagawa, Henry Jay Forman, Jinah Choi, Mónica Medina
    Abstract:

    Hyperthermic stress is known to trigger the loss of unicellular algae from a number of symbiotic cnidarians, a phenomenon commonly referred to as bleaching. Oxidative and nitrosative stress have been suggested to play a major role during the process of bleaching, however the underlying molecular mechanisms are still poorly understood. In animals, the intracellular tripeptide glutathione (GSH) is involved in antioxidant defense, redox homeostasis and intracellular redox signaling. Therefore, we tested the hypothesis that hyperthermal stress-induced bleaching in Aiptasia pallida, a model for symbiotic cnidarians, results in increased levels of GSH synthesis. We report the cDNA sequence and functional analysis of the catalytic subunit of Glutamate-Cysteine Ligase (GCLC), which catalyzes the rate-limiting step in GSH biosynthesis. In a time-series experiment, both GCLC gene expression and total GSH levels increased 4- and 1.5-fold, respectively, in response to hyperthermal stress. These results suggest that hyperthermal stress triggers adaptive increases in intracellular GSH biosynthesis in cnidarians as a protective response to oxidative/ nitrosative stress. Our results show the conserved function of GCLC and GSH across animals while placing a new perspective on the role of GSH in redox signaling during cnidarian bleaching.

  • shp 1 inhibition by 4 hydroxynonenal activates jun n terminal kinase and Glutamate Cysteine Ligase
    American Journal of Respiratory Cell and Molecular Biology, 2008
    Co-Authors: Alessandra Rinna, Henry Jay Forman
    Abstract:

    4-Hydroxy-2-nonenal (HNE), a major lipid peroxidation product, is toxic at high concentrations, but at near-physiological concentrations it induces detoxifying enzymes. Previous data established that in human bronchial epithelial (HBE1) cells, both genes for Glutamate Cysteine Ligase (GCL) are induced by HNE through the c-Jun N-terminal kinase (JNK) pathway. The protein-tyrosine phosphatase SH2 domain containing phosphatase-1 (SHP-1) is thought to play a role as a negative regulator of cell signaling, and has been implicated as such in the JNK pathway. In the present study, SHP-1 was demonstrated to contribute to HNE-induced-gclc expression via regulation of the JNK pathway in HBE1 cells. Treatment of HBE1 cells with HNE induced phosphorylation of mitogen-activated protein kinase kinase 4 (MKK4), JNK, and c-Jun. HNE was able to inhibit protein tyrosine phosphatase activity of SHP-1 through increased degradation of the protein. Furthermore, transfection with small interference RNA SHP-1 showed an enhancement of JNK and c-Jun phosphorylation, but not of MKK4, leading to increased gclc expression. These results demonstrate that SHP-1 plays a role as a negative regulator of the JNK pathway and that HNE activated the JNK pathway by inhibiting SHP-1. Thus, SHP-1 acts as a sensor for HNE and is responsible for an important adaptive response to oxidative stress.

Timothy P Dalton - One of the best experts on this subject based on the ideXlab platform.

  • Early onset senescence occurs when fibroblasts lack the Glutamate-Cysteine Ligase modifier subunit.
    Free Radical Biology and Medicine, 2009
    Co-Authors: Ying Chen, Vasilis Vasiliou, Howard G Shertzer, Elisabet Johansson, Timothy P Dalton
    Abstract:

    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.

  • Curcumin, quercetin, and tBHQ modulate glutathione levels in astrocytes and neurons: importance of the Glutamate Cysteine Ligase modifier subunit
    Journal of Neurochemistry, 2009
    Co-Authors: Suzie Lavoie, Pascal Steullet, René Gysin, Michel Cuenod, Timothy P Dalton, Ying Chen, Kim Q.
    Abstract:

    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.

  • Interaction between the catalytic and modifier subunits of Glutamate-Cysteine Ligase
    Biochemical Pharmacology, 2007
    Co-Authors: Yi Yang, Scott N. Schneider, Howard G Shertzer, Elisabet Johansson, Ying Chen, Timothy P Dalton
    Abstract:

    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.

  • Glutamate Cysteine Ligase Catalysis DEPENDENCE ON ATP AND MODIFIER SUBUNIT FOR REGULATION OF TISSUE GLUTATHIONE LEVELS
    Journal of Biological Chemistry, 2005
    Co-Authors: Ying Chen, Scott N. Schneider, Howard G Shertzer, Timothy P Dalton
    Abstract:

    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.

  • 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, 2002
    Co-Authors: Yi Yang, Howard G Shertzer, Ying Chen, Matthew Z Dieter, Daniel W Nebert, Timothy P Dalton
    Abstract:

    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.

Collin C White - One of the best experts on this subject based on the ideXlab platform.

  • Rapid activation of Glutamate Cysteine Ligase following oxidative stress
    Journal of Biological Chemistry, 2010
    Co-Authors: Cecile M. Krejsa, Collin C White, Jeffrey A Ledbetter, Gary L. Schieven, Christopher C Franklin, Terrance J Kavanagh
    Abstract:

    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.

  • Modulating GSH Synthesis Using Glutamate Cysteine Ligase Transgenic and Gene-Targeted Mice
    Drug Metabolism Reviews, 2008
    Co-Authors: Dianne Botta, Lisa A. Mcconnachie, Portia Vliet-gregg, Monica B. Mcgrath, Isaac Mohar, Collin C White, Terrance J Kavanagh
    Abstract:

    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.

  • Structure, function, and post-translational regulation of the catalytic and modifier subunits of Glutamate Cysteine Ligase.
    Molecular aspects of medicine, 2008
    Co-Authors: Christopher C Franklin, Isaac Mohar, Collin C White, Donald S Backos, Henry Jay Forman, Terrance J Kavanagh
    Abstract:

    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.

  • Optimization and application of Glutamate Cysteine Ligase measurement in wildlife species
    Ecotoxicology and Environmental Safety, 2008
    Co-Authors: Hongmei Wu, John P. Isanhart, Tobias J. Mcbride, Collin C White, Terrance J Kavanagh, Michael J Hooper
    Abstract:

    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.

  • Glutamate Cysteine Ligase modifier subunit deficiency and gender as determinants of acetaminophen-Induced hepatotoxicity in mice
    Toxicological Sciences, 2007
    Co-Authors: Lisa A. Mcconnachie, Francesca N. Hudson, Sam Chatterton-kirchmeier, Robert H. Pierce, Isaac Mohar, Collin C White, Carolina Fernandez, Warren C Ladiges, Carol B Ware, Terrance J Kavanagh
    Abstract:

    The analgesic and antipyretic drug acetaminophen (APAP) is bioactivated to the reactive intermediate N-acetyl-p-benzoquinoneimine, which is scavenged by glutathione (GSH). APAP overdose can deplete GSH leading to the accumulation of APAP-protein adducts and centrilobular necrosis in the liver. N-acetylCysteine (NAC), a Cysteine prodrug and GSH precursor, is often given as a treatment for APAP overdose. The rate-limiting step in GSH biosynthesis is catalyzed by Glutamate Cysteine Ligase (GCL) a heterodimer composed of catalytic and modifier (GCLM) subunits. Previous studies have indicated that GCL activity is likely to be an important determinant of APAP toxicity. In this study, we investigated APAP toxicity, and NAC or GSH ethyl ester (GSHee)-mediated rescue in mice with normal or compromised GCLM expression. Gclm wild-type, heterozygous, and null mice were administered APAP (500 mg/kg) alone, or immediately following NAC (800 mg/kg) or GSHee (168 mg/kg), and assessed for hepatotoxicity 6 h later. APAP caused GSH depletion in all mice. Gclm null and heterozygous mice exhibited more extensive hepatic damage compared to wild-type mice as assessed by serum alanine aminotransferase activity and histopathology. Additionally, male Gclm wild-type mice demonstrated greater APAP-induced hepatotoxicity than female wild-type mice. Cotreatment with either NAC or GSHee mitigated the effects of APAP in Gclm wild-type and heterozygous mice, but not in Gclm null mice. Collectively, these data reassert the importance of GSH in protection against APAP-induced hepatotoxicity, and indicate critical roles for GCL activity and gender in APAP-induced liver damage in mice.

Dale A Dickinson - One of the best experts on this subject based on the ideXlab platform.

  • transforming growth factor β suppresses Glutamate Cysteine Ligase gene expression and induces oxidative stress in a lung fibrosis model
    Free Radical Biology and Medicine, 2012
    Co-Authors: Ruiming Liu, Terrance J Kavanagh, Dale A Dickinson, Praveen K Vayalil, Carol A Ballinger, Wen Tan Huang, Suqing Wang, Qiana L Matthews, Edward M Postlethwait
    Abstract:

    The concentration of glutathione (GSH), the most abundant intracellular free thiol and an important antioxidant, is decreased in the lung in both fibrotic diseases and experimental fibrosis models. The underlying mechanisms and biological significance of GSH depletion, however, remain unclear. Transforming growth factor β (TGF-β) is the most potent and ubiquitous profibrogenic cytokine and its expression is increased in almost all fibrotic diseases. In this study, we show that increasing TGF-β1 expression in mouse lung to a level comparable to those found in lung fibrotic diseases by intranasal instillation of AdTGF-β1(223/225), an adenovirus expressing constitutively active TGF-β1, suppressed the expression of both catalytic and modifier subunits of Glutamate-Cysteine Ligase (GCL), the rate-limiting enzyme in de novo GSH synthesis, decreased GSH concentration, and increased protein and lipid peroxidation in mouse lung. Furthermore, we show that increasing TGF-β1 expression activated JNK and induced activating transcription factor 3, a transcriptional repressor involved in the regulation of the catalytic subunit of GCL, in mouse lung. Control virus (AdDL70-3) had no significant effect on any of these parameters, compared to saline-treated control. Concurrent with GSH depletion, TGF-β1 induced lung epithelial apoptosis and robust pulmonary fibrosis. Importantly, lung GSH levels returned to normal, whereas fibrosis persisted at least 21 days after TGF-β1 instillation. Together, the data suggest that increased TGF-β1 expression may contribute to the GSH depletion observed in pulmonary fibrosis diseases and that GSH depletion may be an early event in, rather than a consequence of, fibrosis development.

  • Nitric oxide-induced resistance to hydrogen peroxide stress is a Glutamate Cysteine Ligase activity-dependent process
    Free Radical Biology and Medicine, 2005
    Co-Authors: Lisa A. Ridnour, Iman M. Ahmad, Mitchell C. Coleman, Prahbat C. Goswami, Clayton R. Hunt, Dale A Dickinson, Henry Jay Forman, Jinah Choi, Douglas R. Spitz
    Abstract:

    Nitric oxide (NO) is a reactive nitrogen species known to be involved in cytotoxic processes. Cells respond to cytotoxic injury by stress response induction leading to the development of cellular resistance. This report describes an NO-induced stress response in Chinese hamster fibroblasts (HA1), which leads to glutathione synthesis-dependent resistance to H2O2-mediated oxidative stress. The development of resistance to H2O2 was completely abolished by the inhibition of Glutamate Cysteine Ligase (GCL) during the first 8 h of recovery after NO exposure. Altered thiol metabolism was observed immediately after NO exposure as demonstrated by up to 75% decrease in intracellular thiol pools (glutathione, γ-glutamylCysteine, and Cysteine), which then reaccumulated during the NO-mediated development of resistance. Immunoreactive protein and activity associated with GCL decreased immediately after exposure to NO and then reaccumulated during the development of resistance to H2O2 challenge. Moreover, compared to N2 controls the activity levels of GCL in NO-exposed cells increased approximately twofold 24 h after H2O2 challenge. These results demonstrate that NO exposure is capable of inducing an adaptive response to H2O2-mediated oxidative stress in mammalian cells, which involves alterations in thiol metabolism and is dependent upon glutathione synthesis and increased GCL activity.

  • Human Glutamate Cysteine Ligase gene regulation through the electrophile response element.
    Free Radical Biology and Medicine, 2004
    Co-Authors: Dale A Dickinson, Erin K. Arnold, Hongqiao Zhang, Anna-liisa Levonen, Douglas R Moellering, Victor M. Darley-usmar, Henry Jay Forman
    Abstract:

    Glutathione (GSH) is the primary nonprotein thiol in the cell. It has many important roles in cell function, including regulating redox-dependent signal transduction pathways. The content of GSH within the cell varies with stress. In many cases, a process involving GSH synthesis results in adaptation to subsequent stressors. Sustained increases in GSH content are controlled primarily through induction of two genes, Gclc and Gclm, leading to the synthesis of the rate-limiting enzyme for GSH synthesis, Glutamate Cysteine Ligase. Each of these genes in humans has a number of putative enhancer elements in their promoters. Overall, the most important element in both Gclc and Gclm expression is the electrophile response element. We review the evidence that has led to this conclusion and the implications for the redox-dependent regulation of this critical intracellular antioxidant.

  • Variable regulation of Glutamate Cysteine Ligase subunit proteins affects glutathione biosynthesis in response to oxidative stress
    Archives of Biochemistry and Biophysics, 2004
    Co-Authors: David M Krzywanski, Karen E. Iles, Amanda F. Wigley, Terrance J Kavanagh, Dale A Dickinson, Christopher C Franklin, Henry Jay Forman
    Abstract:

    Glutamate Cysteine Ligase (GCL), composed of a catalytic (GCLC) and modulatory (GCLM) subunit, catalyzes the first step of glutathione (GSH) biosynthesis. Using 4-hydroxy-2-nonenal (4HNE), 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), and tertiary-butylhydroquinone (tBHQ) as models of oxidative stress which are known to work through different mechanisms, we measured changes in cellular GSH, GCL mRNA, and GCL protein. 4HNE and tBHQ treatments increased cellular GSH levels, while DMNQ exposure depleted GSH. Furthermore, changes in the two GCL mRNAs largely paralleled changes in the GCL proteins; however, the magnitudes differed, suggesting some form of translational control. The molar ratio of GCLC:GCLM ranged from 3:1 to 17:1 in control human bronchial epithelial (HBE1) cells and all treatments further increased this ratio. Data from several mouse tissues show molar ratios of GCLC:GCLM that range from 1:1 to 10:1 in support of these findings. These data demonstrate that alterations in cellular GSH are clearly correlated with GCLC to a greater extent than GCLM. Surprisingly, both control HBE1 cells and some mouse tissues have more GCLC than GCLM and GCLM increases to a much lesser extent than GCLC, suggesting that the regulatory role of GCLM is minimal under physiologically relevant conditions of oxidative stress.

  • curcumin alters epre and ap 1 binding complexes and elevates Glutamate Cysteine Ligase gene expression
    The FASEB Journal, 2003
    Co-Authors: Dale A Dickinson, Karen E. Iles, Hongqiao Zhang, Volker Blank, Henry Jay Forman
    Abstract:

    Dietary use of curcumin, the active component of tumeric, one of the most widely used spices, is linked to several beneficial health effects, although the underlying molecular mechanisms remain largely unknown. Correlations have been established between curcumin exposure and increases in enzymes for glutathione synthesis, particularly Glutamate-Cysteine Ligase (GCL), and metabolism as well as glutathione content, suggesting the eliciting of an adaptive response to stress. In this study, using HBE1 cells, we found that the mechanism of curcumin-induced GCL elevation occurred via transcription of the two Gcl genes. Gcl transcription has been shown in several systems to be mediated through binding of transcription factor complexes to TRE and EpRE elements. Studies herein showed that curcumin caused modest but sustained increases in binding of proteins to DNA sequences for both cis elements but, more importantly, altered the compositions and nuclear content of proteins in these complexes. Curcumin exposure increased JunD and c-Jun content in AP-1 complexes and increased JunD while decreasing MafG/MafK in EpRE complexes. Thus, the beneficial effects elicited by curcumin appear to be due to changes in the pool of transcription factors that compose EpRE and AP-1 complexes, affecting gene expression of GCL and other phase II enzymes.