Glutamate Cysteine Ligase Holoenzyme Research Articles

Post-translational Activation of Glutamate Cysteine Ligase with Dimercaprol: A NOVEL MECHANISM OF INHIBITING NEUROINFLAMMATION IN VITRO

Neuroinflammation and oxidative stress are hallmarks of various neurological diseases. However, whether and how the redox processes control neuroinflammation is incompletely understood. We hypothesized that increasing cellular glutathione (GSH) levels would inhibit neuroinflammation. A series of thiol compounds were identified to elevate cellular GSH levels by a novel approach (i.e. post-translational activation of glutamate cysteine ligase (GCL), the rate-limiting enzyme in GSH biosynthesis). These small thiol-containing compounds were examined for their ability to increase intracellular GSH levels in a murine microglial cell line (BV2), of which dimercaprol (2,3-dimercapto-1-propanol (DMP)) was found to be the most effective compound. DMP increased GCL activity and decreased LPS-induced production of pro-inflammatory cytokines and inducible nitric-oxide synthase induction in BV2 cells in a concentration-dependent manner. The ability of DMP to elevate GSH levels and attenuate LPS-induced pro-inflammatory cytokine production was inhibited by buthionine sulfoximine, an inhibitor of GCL. DMP increased the expression of GCL holoenzyme without altering the expression of its subunits or Nrf2 target proteins (NQO1 and HO-1), suggesting a post-translational mechanism. DMP attenuated LPS-induced MAPK activation in BV2 cells, suggesting the MAPK pathway as the signaling mechanism underlying the effect of DMP. Finally, the ability of DMP to increase GSH via GCL activation was observed in mixed cerebrocortical cultures and N27 dopaminergic cells. Together, the data demonstrate a novel mechanism of GSH elevation by post-translational activation of GCL. Post-translational activation of GCL offers a novel targeted approach to control inflammation in chronic neuronal disorders associated with impaired adaptive responses.

Glycation of Glutamate Cysteine Ligase by 2-Deoxy-d-Ribose and its Potential Impact on Chemoresistance in Glioblastoma

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.

Low-level domoic acid protects mouse cerebellar granule neurons from acute neurotoxicity: role of glutathione.

Domoic acid (DomA) is a potent marine neurotoxin. By activating α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid/kainate receptors, DomA induces oxidative stress-mediated apoptotic cell death in neurons. The effect of prolonged (10 days) exposure to a low, nontoxic concentration (5nM) of DomA on acute (intermediate concentration) neurotoxicity of this toxin was investigated in cerebellar granule neurons (CGNs) from wild-type mice and mice lacking the glutamate cysteine ligase (GCL) modifier subunit (Gclm (/)). CGNs from Gclm (/) mice have very low glutathione (GSH) levels and are very sensitive to DomA toxicity. In CGNs from wild-type mice, prolonged exposure to 5nM DomA did not cause any overt toxicity but reduced oxidative stress-mediated apoptotic cell death induced by exposure to an intermediate concentration (100nM for 24h) of DomA. This protection was not observed in CGNs from Gclm (/) mice. Prolonged DomA exposure increased GSH levels in CGNs of wild-type but not Gclm (/) mice. Levels of GCLC (the catalytic subunit of GCL) protein and mRNA were increased in CGNs of both mouse strains, whereas levels of GCLM protein and mRNA, activity of GCL, and levels of GCL holoenzyme were only increased in CGNs of wild-type mice. Chronic DomA exposure also protected wild-type CGNs from acute toxicity of other oxidants. The results indicate that CGNs from Gclm (/) mice, which are already more sensitive to DomA toxicity, are unable to upregulate their GSH levels. As Gclm (/) mice may represent a model for a common human polymorphism in GCLM, such individuals may be at particular risk for DomA-induced neurotoxicity.

Circadian Regulation of Glutathione Levels and Biosynthesis in Drosophila melanogaster

Circadian clocks generate daily rhythms in neuronal, physiological, and metabolic functions. Previous studies in mammals reported daily fluctuations in levels of the major endogenous antioxidant, glutathione (GSH), but the molecular mechanisms that govern such fluctuations remained unknown. To address this question, we used the model species Drosophila, which has a rich arsenal of genetic tools. Previously, we showed that loss of the circadian clock increased oxidative damage and caused neurodegenerative changes in the brain, while enhanced GSH production in neuronal tissue conferred beneficial effects on fly survivorship under normal and stress conditions. In the current study we report that the GSH concentrations in fly heads fluctuate in a circadian clock-dependent manner. We further demonstrate a rhythm in activity of glutamate cysteine ligase (GCL), the rate-limiting enzyme in glutathione biosynthesis. Significant rhythms were also observed for mRNA levels of genes encoding the catalytic (Gclc) and modulatory (Gclm) subunits comprising the GCL holoenzyme. Furthermore, we found that the expression of a glutathione S-transferase, GstD1, which utilizes GSH in cellular detoxification, significantly fluctuated during the circadian day. To directly address the role of the clock in regulating GSH-related rhythms, the expression levels of the GCL subunits and GstD1, as well as GCL activity and GSH production were evaluated in flies with a null mutation in the clock genes cycle and period. The rhythms observed in control flies were not evident in the clock mutants, thus linking glutathione production and utilization to the circadian system. Together, these data suggest that the circadian system modulates pathways involved in production and utilization of glutathione.

Glutathione-Deficient Mice Are Susceptible to TCDD-Induced Hepatocellular Toxicity but Resistant to Steatosis

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) generates both hepatocellular injury and steatosis, processes that involve oxidative stress. Herein, we evaluated the role of the antioxidant glutathione (GSH) in TCDD-induced hepatotoxicity. Glutamate-cysteine ligase (GCL), comprising catalytic (GCLC) and modifier (GCLM) subunits, is rate limiting in de novo GSH biosynthesis; GCLM maintains GSH homeostasis by optimizing the catalytic efficiency of GCL holoenzyme. Gclm(-/-) transgenic mice exhibit 10-20% of normal tissue GSH levels. Gclm(-/-) and Gclm(+/+) wild-type (WT) female mice received TCDD for 3 consecutive days and were then examined 21 days later. As compared with WT littermates, Gclm(-/-) mice were more sensitive to TCDD-induced hepatocellular toxicity, exhibiting lower reduction potentials for GSH, lower ATP levels, and elevated levels of plasma glutamic oxaloacetic transaminase (GOT) and γ-glutamyl transferase (GGT). However, the histopathology showed that TCDD-mediated steatosis, which occurs in WT mice, was absent in Gclm(-/-) mice. This finding was consistent with cDNA microarray expression analysis, revealing striking deficiencies in lipid biosynthesis pathways in Gclm(-/-) mice; qrt-PCR analysis confirmed that Gclm(-/-) mice are deficient in expression of several lipid metabolism genes including Srebp2, Elovl6, Fasn, Scd1/2, Ppargc1a, and Ppara. We suggest that whereas GSH protects against TCDD-mediated hepatocellular damage, GSH deficiency confers resistance to TCDD-induced steatosis due to impaired lipid metabolism.

Posttranslational modification and regulation of glutamate–cysteine ligase by the α,β-unsaturated aldehyde 4-hydroxy-2-nonenal

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.

Rapid Activation of Glutamate Cysteine Ligase following Oxidative Stress

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.

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.

Initial Characterization of the Glutamate-Cysteine Ligase Modifier Subunit Gclm(−/−) Knockout Mouse: NOVEL MODEL SYSTEM FOR A SEVERELY COMPROMISED OXIDATIVE STRESS RESPONSE

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.

4-hydroxynonenal induces glutamate cysteine ligase through JNK in HBE1 cells.

Glutathione is the most abundant non-protein thiol in the cell, with roles in cell cycle regulation, detoxification of xenobiotics, and maintaining the redox tone of the cell. The glutathione content is controlled at several levels, the most important being the rate of de novo synthesis, which is mediated by two enzymes, glutamate cysteine ligase (GCL), and glutathione synthetase (GS), with GCL being rate-limiting generally. The GCL holoenzyme consists of a catalytic (GCLC) and a modulatory (GCLM) subunit, which are encoded by separate genes. In the present study, the signaling mechanisms leading to de novo synthesis of GSH in response to physiologically relevant concentrations of 4-hydroxy-2-nonenal (4HNE), an endproduct of lipid peroxidation, were investigated. We demonstrated that exposure to 4HNE resulted in increased content of both Gcl mRNAs, both GCL subunits, phosphorylated JNK1 and c-Jun proteins, as well as Gcl TRE sequence-specific AP-1 binding activity. These increases were attenuated by pretreating the cells with a novel membrane-permeable JNK pathway inhibitor, while chemical inhibitors of the p38 or ERK pathways were ineffective. These data reveal that de novo GSH biosynthesis in response to 4HNE signals through the JNK pathway and suggests a major role for AP-1 driven expression of both Gcl genes in HBE1 cells.