Collin C. White

Structure, function, and post-translational regulation of the catalytic and modifier subunits of glutamate cysteine ligase

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.

Fluorescence-based microtiter plate assay for glutamate–cysteine ligase activity

Glutamate-cysteine ligase (GCL; also known as gamma-glutamylcysteine synthetase) is the rate-limiting enzyme in glutathione (GSH) synthesis. Traditional assays for the activity of this enzyme are based either on coupled reactions with other enzymes or on high-performance liquid chromatography (HPLC) assessment of gamma-glutamylcysteine (gamma-GC) product formation. We took advantage of the reaction of naphthalene dicarboxaldehyde (NDA) with GSH or gamma-GC to form cyclized products that are highly fluorescent. Hepa-1 cells which were designed to overexpress mouse GCL and mouse liver homogenates were used to evaluate and compare the utility of the NDA method with an assay based on monobromobimane derivatization and HPLC analysis with fluorescence detection. Excellent agreement was found between GCL activities measured by HPLC and NDA-microtiter plate analyses. This assay should be useful for high-throughput GCL activity analyses.

Neurotoxicity of domoic acid in cerebellar granule neurons in a genetic model of glutathione deficiency

This study investigated the role of cellular antioxidant defense mechanisms in modulating the neurotoxicity of domoic acid (DomA), by using cerebellar granule neurons (CGNs) from mice lacking the modifier subunit of glutamate-cysteine ligase (Gclm). Glutamate-cysteine ligase (Glc) catalyzes the first and rate-limiting step in glutathione (GSH) biosynthesis. CGNs from Gclm (-/-) mice have very low levels of GSH and are 10-fold more sensitive to DomA-induced toxicity than CGNs from Gclm (+/+) mice. GSH ethyl ester decreased, whereas the Gcl inhibitor buthionine sulfoximine increased DomA toxicity. Antagonists of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate receptors and of N-methyl-d-aspartate (NMDA) receptors blocked DomA toxicity, and NMDA receptors were activated by DomA-induced l-glutamate release. The differential susceptibility of CGNs to DomA toxicity was not due to a differential expression of ionotropic glutamate receptors, as evidenced by similar calcium responses and l-glutamate release in the two genotypes. A calcium chelator and several antioxidants antagonized DomA-induced toxicity. DomA caused a rapid decrease in cellular GSH, which preceded toxicity, and the decrease was primarily due to DomA-induced GSH efflux. DomA also caused an increase in oxidative stress as indicated by increases in reactive oxygen species and lipid peroxidation, which was subsequent to GSH efflux. Astrocytes from both genotypes were resistant to DomA toxicity and presented a diminished calcium response to DomA and a lack of DomA-induced l-glutamate release. Because polymorphisms in the GCLM gene in humans are associated with low GSH levels, such individuals, as well as others with genetic conditions or environmental exposures that lead to GSH deficiency, may be more susceptible to DomA-induced neurotoxicity.

TGFβ1-induced suppression of glutathione antioxidant defenses in hepatocytes: caspase-dependent post-translational and caspase-independent transcriptional regulatory mechanisms

TGFβ1‐induced hepatocyte apoptosis involves the production of reactive oxygen species. An effective cellular defense mechanism against oxidative stress is the tripeptide glutathione (GSH), and the rate‐limiting step in GSH biosynthesis is catalyzed by the heterodimeric holoenzyme glutamate cysteine ligase (GCL). Here, we demonstrate that TGFβ1‐induced apoptosis in the TAMH murine hepatocyte cell line is accompanied by both the cleavage and loss of the catalytic subunit of GCL (GCLC) and the down‐regulation of GCLC gene expression resulting in a reduction in GCL activity and depletion of intracellular GSH. TGFβ1‐induced apoptosis is also accompanied by a reduction in Bcl‐XL, an effect that may facilitate TGFβ1‐induced apoptosis as Bcl‐XL overexpression inhibits TGFβ1‐induced caspase activation and cell death. Interestingly, Bcl‐XL overexpression prevents TGFβ1‐induced cleavage of GCLC protein but not down‐regulation of GCLC mRNA. Furthermore, TGFβ1‐induced down‐regulation of GCLC mRNA is prevented by inhibition of histone deacetylase activity, suggesting that this is an active repression of GCLC gene transcription. These findings suggest that the suppression of GSH antioxidant defenses associated with the caspase‐dependent cleavage of GCLC protein, caspase‐independent suppression of GCLC gene expression, and depletion of intracellular GSH may play a role in enhancing TGFβ1‐induced oxidative stress and potentiating apoptotic cell death.

Induction of glutamate-cysteine ligase (γ-glutamylcysteine synthetase) in the brains of adult female mice subchronically exposed to methylmercury

Methylmercury (MeHg) is widely known for its potent neurotoxic properties. One proposed mechanism of action of MeHg relates to its high affinity for sulfhydryl groups, especially those found on glutathione (GSH) and proteins. Previous studies have shown that acute MeHg exposure results in an increase in the mRNA for the rate-limiting enzyme in GSH synthesis, glutamate-cysteine ligase (GLCL) (also known as gamma-glutamylcysteine synthetase). In this study, we evaluated the effects of subchronic (12-week) MeHg exposure at 0, 3 or 10 ppm in the drinking water on GSH levels, GLCL catalytic (GLCLC) and regulatory subunit mRNA and protein levels, and GLCL activity in brain, liver and kidney tissue of C57B1/6 female mice. Contrary to previous findings in rats, there were no changes in GSH concentration in any of the tissues examined. However, there was an increase in GLCLC protein in the brain, which was accompanied by a 30% increase in GLCL activity. We conclude that up-regulation of GSH synthetic capacity in the brains of mice is a sensitive biomarker of subchronic MeHg exposure.

Mitochondrial‐targeted peptide rapidly improves mitochondrial energetics and skeletal muscle performance in aged mice

Mitochondrial dysfunction plays a key pathogenic role in aging skeletal muscle resulting in significant healthcare costs in the developed world. However, there is no pharmacologic treatment to rapidly reverse mitochondrial deficits in the elderly. Here, we demonstrate that a single treatment with the mitochondrial‐targeted peptide SS‐31 restores in vivo mitochondrial energetics to young levels in aged mice after only one hour. Young (5 month old) and old (27 month old) mice were injected intraperitoneally with either saline or 3 mg kg−1 of SS‐31. Skeletal muscle mitochondrial energetics were measured in vivo one hour after injection using a unique combination of optical and 31P magnetic resonance spectroscopy. Age‐related declines in resting and maximal mitochondrial ATP production, coupling of oxidative phosphorylation (P/O), and cell energy state (PCr/ATP) were rapidly reversed after SS‐31 treatment, while SS‐31 had no observable effect on young muscle. These effects of SS‐31 on mitochondrial energetics in aged muscle were also associated with a more reduced glutathione redox status and lower mitochondrial H2O2 emission. Skeletal muscle of aged mice was more fatigue resistant in situ one hour after SS‐31 treatment, and eight days of SS‐31 treatment led to increased whole‐animal endurance capacity. These data demonstrate that SS‐31 represents a new strategy for reversing age‐related deficits in skeletal muscle with potential for translation into human use.

Caspase-3-Dependent Cleavage of the Glutamate-L-Cysteine Ligase Catalytic Subunit during Apoptotic Cell Death.

Apoptotic cell death is usually accompanied by activation of a family of cysteine proteases termed caspases. Caspases mediate the selective proteolysis of multiple cellular targets often resulting in the disruption of survival pathways. Intracellular levels of the antioxidant glutathione (GSH) are an important determinant of cellular susceptibility to apoptosis. The rate-limiting step in GSH biosynthesis is mediated by glutamate-L-cysteine ligase (GCL), a heterodimeric enzyme consisting of a catalytic (GCLC) and a modifier (GCLM) subunit. In this report we demonstrate that GCLC is a direct target for caspase-mediated cleavage in multiple models of apoptotic cell death. Mutational analysis revealed that caspase-mediated cleavage of GCLC occurs at Asp(499) within the sequence AVVD(499)G. GCLC cleavage occurs upstream of Cys(553), which is thought to be important for association with GCLM. GCLC cleavage is accompanied by a rapid loss of intracellular GSH due to caspase-mediated extrusion of GSH from the cell. However, while GCLC cleavage is dependent on caspase-3, GSH extrusion occurs by a caspase-3-independent mechanism. Our identification of GCLC as a target for caspase-3-dependent cleavage during apoptotic cell death suggests that this post-translational modification may represent a novel mechanism for regulating GSH biosynthesis during apoptosis.

Gonadotropin regulation of glutathione synthesis in the rat ovary

Glutathione (GSH), an antioxidant and conjugator of electrophilic toxicants, prevents toxicant-mediated destruction of ovarian follicles and oocytes. Ovarian GSH has previously been shown to change with estrous cycle stage in rats, suggesting that the gonadotropin hormones may regulate ovarian GSH synthesis. The present studies tested the hypotheses that [1] estrous cycle-related changes in ovarian GSH result from cyclic changes in protein and mRNA expression of the rate-limiting enzyme in GSH synthesis, glutamate cysteine ligase (GCL, also called gamma-glutamylcysteine synthetase), and [2] that these changes result from gonadotropin-mediated regulation of GCL subunit expression. In the first experiment, ovaries were harvested from cycling adult female rats on each stage of the estrous cycle. In the second experiment immature female rats were injected with pregnant mares serum gonadotropin (PMSG) to stimulate follicular development or with vehicle and killed 8, 24, or 48 h later. In both experiments the ovaries were harvested for [1] total GSH assay, [2] Western analysis for GCL catalytic (GCLc) and regulatory (GCLm) subunit protein levels, or [3] Northern analysis for Gclc and Gclm mRNA levels. Ovarian GSH concentrations and Gclc and Gclm mRNA levels, but not GCL subunit protein levels, varied significantly with estrous cycle stage. PMSG administration significantly increased ovarian GSH concentrations 24 and 48 h later. GCLm protein levels increased significantly at 24 h and 48 h following PMSG. GCLc protein levels did not increase significantly following PMSG. Gcl subunit mRNA levels were not significantly increased at any time point by the planned ANOVA; however, an increase in Gelc at 48 h was identified by t-testing. These results support the hypothesis that gonadotropins regulate ovarian GSH synthesis by modulating GCL subunit expression.

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.

Acetaminophen-induced liver injury is attenuated in male glutamate-cysteine ligase transgenic mice.

Acetaminophen overdose is a leading cause of drug-related acute liver failure in the United States. Glutathione, a tripeptide antioxidant protects cells against oxidative damage from reactive oxygen species and plays a crucial role in the detoxification of xenobiotics, including acetaminophen. Glutathione is synthesized in a two-step enzymatic reaction. Glutamate-cysteine ligase carries out the rate-limiting and first step in glutathione synthesis. We have generated C57Bl/6 mice that conditionally overexpress glutamate-cysteine ligase, and report here their resistance to acetaminophen-induced liver injury. Indices of liver injury included histopathology and serum alanine aminotransferase activity. Male transgenic mice induced to overexpress glutamate-cysteine ligase exhibited resistance to acetaminophen-induced liver injury when compared with acetaminophen-treated male mice carrying, but not expressing glutamate-cysteine ligase transgenes, or to female glutamate-cysteine ligase transgenic mice. We conclude that glutamate-cysteine ligase activity is an important factor in determining acetaminophen-induced liver injury in C57Bl/6 male mice. Because people are known to vary in their glutamate-cysteine ligase activity, this enzyme may also be an important determinant of sensitivity to acetaminophen-induced liver injury in humans.