Christopher C. Franklin

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.

Disruption of redox homeostasis in tumor necrosis factor-induced apoptosis in a murine hepatocyte cell line.

Tumor necrosis factor (TNF) is a mediator of the acute phase response in the liver and can initiate proliferation and cause cell death in hepatocytes. We investigated the mechanisms by which TNF causes apoptosis in hepatocytes focusing on the role of oxidative stress, antioxidant defenses, and mitochondrial damage. The studies were conducted in cultured AML12 cells, a line of differentiated murine hepatocytes. As is the case for hepatocytes in vivo, AML12 cells were not sensitive to cell death by TNF alone, but died by apoptosis when exposed to TNF and a small dose of actinomycin D (Act D). Morphological signs of apoptosis were not detected until 6 hours after the treatment and by 18 hours approximately 50% of the cells had died. Exposure of the cells to TNF+Act D did not block NFkappaB nuclear translocation, DNA binding, or its overall transactivation capacity. Induction of apoptosis was characterized by oxidative stress indicated by the loss of NAD(P)H and glutathione followed by mitochondrial damage that included loss of mitochondrial membrane potential, inner membrane structural damage, and mitochondrial condensation. These changes coincided with cytochrome C release and the activation of caspases-8, -9, and -3. TNF-induced apoptosis was dependent on glutathione levels. In cells with decreased levels of glutathione, TNF by itself in the absence of transcriptional blocking acted as an apoptotic agent. Conversely, the antioxidant alpha-lipoic acid, that protected against the loss of glutathione in cells exposed to TNF+Act D completely prevented mitochondrial damage, caspase activation, cytochrome C release, and apoptosis. The results demonstrate that apoptosis induced by TNF+Act D in AML12 cells involves oxidative injury and mitochondrial damage. As injury was regulated to a larger extent by the glutathione content of the cells, we suggest that the combination of TNF+Act D causes apoptosis because Act D blocks the transcription of genes required for antioxidant defenses.

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.

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.

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.

Cell culture model for acetaminophen-induced hepatocyte death in vivo

Overdose of the popular, and relatively safe, analgesic acetaminophen (N-acetyl-p-aminophenol, APAP, paracetamol) can produce a fatal centrilobular liver injury. APAP-induced cell death was investigated in a differentiated, transforming growth factor alpha (TGFalpha)-overexpressing, hepatocyte cell line and found to occur at concentrations, and over time frames, relevant to clinical overdose situations. Coordinated multiorganellar collapse was evident during APAP-induced cytotoxicity with widespread, yet selective, protein degradation events in vitro. Cellular proteasomal activity was inhibited with APAP treatment but not with the comparatively nonhepatotoxic APAP regioisomer, N-acetyl-m-aminophenol (AMAP). Low concentrations of the proteasome-directed inhibitor MG132 (N-carbobenzoxyl-Leu-Leu-Leucinal) increased chromatin condensation and cellular stress responses preferentially in AMAP-treated cultures, suggesting a contribution of the proteasome in APAP- but not AMAP-mediated cell death. APAP-specific alterations to mitochondria were observed morphologically with evidence of mitochondrial proliferation in vitro. Biochemical alterations to cellular proteolytic events were also found in vivo, including APAP- or AMAP-mediated inhibition of caspase-3 processing. These results indicate that, although retaining some attributes of apoptosis, both APAP- and AMAP-mediated cell death have additional distinctive features consistent with longer term necrosis.

Oxidative Stress and the ER Stress Response in a Murine Model for Early-Stage Alcoholic Liver Disease

Alcoholic liver disease (ALD) is a primary cause of morbidity and mortality in the United States and constitutes a significant socioeconomic burden. Previous work has implicated oxidative stress and endoplasmic reticulum (ER) stress in the etiology of ALD; however, the complex and interrelated nature of these cellular responses presently confounds our understanding of ethanol-induced hepatopathy. In this paper, we assessed the pathological contribution of oxidative stress and ER stress in a time-course mouse model of early-stage ALD. Ethanol-treated mice exhibited significant hepatic panlobular steatosis and elevated plasma ALT values compared to isocaloric controls. Oxidative stress was observed in the ethanol-treated animals through a significant increase in hepatic TBARS and immunohistochemical staining of 4-HNE-modified proteins. Hepatic glutathione (GSH) levels were significantly decreased as a consequence of decreased CBS activity, increased GSH utilization, and increased protein glutathionylation. At the same time, immunoblot analysis of the PERK, IRE1α, ATF6, and SREBP pathways reveals no significant role for these UPR pathways in the etiology of hepatic steatosis associated with early-stage ALD. Collectively, our results indicate a primary pathogenic role for oxidative stress in the early initiating stages of ALD that precedes the involvement of the ER stress response.

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.

Protection of acute myeloblastic leukemia cells against apoptotic cell death by high glutathione and gamma-glutamylcysteine synthetase levels during etoposide-induced oxidative stress

BACKGROUND Etoposide mediates its cytotoxicity by inducing apoptosis. Thus, mechanisms which regulate apoptosis should also affect drug resistance. Oxidants and antioxidants have been shown to participate in the regulation of apoptosis. We were interested in studying whether responsiveness of acute myeloblastic leukemia (AML) cells to etoposide is mediated by oxidative stress and glutathione levels. PATIENTS AND METHODS Two subclones of the OCI/AML-2 cell line which are etoposide-sensitive (ES), and etoposide-resistant (ER), were established by the authors at the University of Oulu, and used as models. Assays for apoptosis included externalization of phosphatidylserine (as evidenced by annexin V binding), and caspase activation as indicated by cleavage of poly(ADP-ribose)polymerase (Western blotting). Peroxide formation was analyzed by flow cytometry. Glutathione and gamma-glutamylcysteine synthetase (gamma-GCS) levels were determined spectrophotometrically and by Western blotting, respectively. RESULTS Etoposide-induced apoptosis was evident 12 hours after treatment in the ES subclone, but was apparent in the ER subclone only after 24 hours. The basal glutathione and gamma-GCS levels were higher in the ER than the ES subclone. Etoposide increased peroxide formation in both subclones after 12-hour exposure. Significant depletion of glutathione was observed in the ES subclone during etoposide exposure, while glutathione levels were maintained in the ER subclone. In neither of the subclones was induction of gamma-GCS observed during 24-hour exposure to etoposide. Furthermore, the catalytic subunit of gamma-GCS was cleaved during apoptosis, concurrent with depletion of intracellular glutathione. When glutathione was depleted by treatment with buthionine sulfoximine, a direct inhibitor of gamma-GCS, the sensitivity to etoposide was increased, particularly in the ER subclone. CONCLUSIONS The results underline the significance of glutathione biosynthesis in the responsiveness of AML cells to etoposide. The molecular mechanisms mediating glutathione depletion during etoposide exposure might include the cleavage of the catalytic subunit of gamma-GCS.

Distinct Nrf1/2-independent mechanisms mediate As3+-induced glutamate-cysteine ligase subunit gene expression in murine hepatocytes

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