Knockout of the mouse glutamate cysteine ligase catalytic subunit (Gclc) gene: embryonic lethal when homozygous, and proposed model for moderate glutathione deficiency when heterozygous

The biosynthesis of reduced glutathione (GSH) is carried out by the enzymes gamma-glutamylcysteine synthetase (GCL) and GSH synthetase. GCL is the rate-limiting step and represents a heterodimeric enzyme comprised of a catalytic subunit (GCLC) and a ("regulatory"), or modifier, subunit (GCLM). The nonhomologous Gclc and Gclm genes are located on mouse chromosomes 9 and 3, respectively. GCLC owns the catalytic activity, whereas GCLM enhances the enzyme activity by lowering the K(m) for glutamate and increasing the K(i) to GSH inhibition. Humans have been identified with one or two defective GCLC alleles and show low GSH levels. As an initial first step toward understanding the role of GSH in cellular redox homeostasis, we have targeted a disruption of the mouse Gclc gene. The Gclc(-/-) homozygous knockout animal dies before gestational day 13, whereas the Gclc(+/-) heterozygote is viable and fertile. The Gclc(+/-) mouse exhibits a gene-dose decrease in the GCLC protein and GCL activity, but only about a 20% diminution in GSH levels and a compensatory increase of approximately 30% in ascorbate-as compared with that in Gclc(+/+) wild-type littermates. These data show a reciprocal action between falling GSH concentrations and rising ascorbate levels. Therefore, the Gclc(+/-) mouse may be a useful genetic model for mild endogenous oxidative stress.     

Redox control of G(1)/S cell cycle regulators during nitric oxide-mediated cell cycle arrest

Redox regulation of cell cycle progression during nitric oxide (NO) mediated cytostasis is not well-understood. In this study, we investigated the role of the intracellular antioxidant glutathione (GSH) in regulating specific signaling events that are associated with NO-mediated cell cycle arrest. Manipulation of intracellular GSH content through pharmacological inhibition of glutamate-cysteine ligase (GCL) indicated that GSH depletion potentiated nitrosative stress, DNA damage, phosphorylation of the tumor suppressor p53 (Ser-18) and upregulation of p21(cip1/waf1) upon NO stimulation. However, we found that neither overexpression of a dominant negative p53 nor pharmacological inhibition of p53 with cyclic pifithrin-alpha (cPFT-alpha) was sufficient to reverse NO-mediated cell cycle arrest or hypophosphorylation of retinoblastoma protein (Rb). We found that the decrease in cyclin D1 levels induced by NO was GSH-sensitive implying that the redox regulation of NO-mediated cytostasis was a multifaceted process and that both p53/p21(cip1/waf1) and p53 independent cyclin D1 pathways were involved. Together, our results demonstrate that GSH serves as an important component of cellular protective mechanisms against NO-derived nitrosative stress to regulate DNA damage checkpoint.     

Cnidarian Primary Cell Culture as a Tool to Investigate the Effect of Thermal Stress at Cellular Level

In the context of global change, symbiotic cnidarians are largely affected by seawater temperature elevation leading to symbiosis breakdown. This process, also called bleaching, is triggered by the dysfunction of the symbiont photosystems causing an oxidative stress and cell death to both symbiont and host cells. In our study, we wanted to elucidate the intrinsic capacity of isolated animal cells to deal with thermal stress in the absence of symbiont. In that aim, we have characterized an animal primary cell culture form regenerating tentacles of the temperate sea anemone Anemonia viridis. We first compared the potential of whole tissue tentacle or separated epidermal or gastrodermal monolayers as tissue sources to settle animal cell cultures. Interestingly, only isolated cells extracted from whole tentacles allowed establishing a viable and proliferative primary cell culture throughout 31 days. The analysis of the expression of tissue-specific and pluripotency markers defined cultivated cells as differentiated cells with gastrodermal origin. The characterization of the animal primary cell culture allowed us to submit the obtained gastrodermal cells to hyperthermal stress (+?5 and +?8 °C) during 1 and 7 days. Though cell viability was not affected at both hyperthermal stress conditions, cell growth drastically decreased. In addition, only a +?8 °C hyperthermia induced a transient increase of antioxidant defences at 1 day but no ubiquitin or carbonylation protein damages. These results demonstrated an intrinsic resistance of cnidarian gastrodermal cells to hyperthermal stress and then confirmed the role of symbionts in the hyperthermia sensitivity leading to bleaching.     

Carbon monoxide produced by heme oxygenase-1 in response to nitrosative stress induces expression of glutamate-cysteine ligase in PC12 cells via activation of phosphatidylinositol 3-kinase and Nrf2 signaling

Induction of heme oxygenase-1 (HO-1) expression has been associated with adaptive cytoprotection against a wide array of toxic insults, but the underlying molecular mechanisms remain largely unresolved. In this study, we investigated the potential role of carbon monoxide (CO), one of the by-products of the HO-1 reaction, in the adaptive survival response to peroxynitrite-induced PC12 cell death. Upon treatment of rat pheochromocytoma (PC12) cells with the peroxynitrite generator 3-morpholinosydnonimine hydrochloride (SIN-1), the cellular GSH level decreased initially, but was gradually restored to the basal level. This was accompanied by increased expression of the catalytic subunit of glutamate-cysteine ligase (GCLC), the rate-limiting enzyme in GSH biosynthesis. The SIN-1-induced GCLC up-regulation was preceded by induction of HO-1 and subsequent CO production. Inhibition of HO activity by zinc protoporphyrin IX or knockdown of HO-1 gene expression by small interfering RNA abrogated the up-regulation of GCLC expression and the subsequent GSH restoration induced by SIN-1. In contrast, additional exposure to the CO-releasing molecule (CO-RM) restored the GSH level previously reduced by inhibition of CO production using zinc protoporphyrin IX. Furthermore, CO-RM treatment up-regulated GCLC expression through activation of Nrf2. The CO-RM-induced activation of Nrf2 was under the control of the phosphatidylinositol 3-kinase/Akt signaling pathway. In conclusion, CO produced by HO-1 rescues PC12 cells from nitrosative stress through induction of GCLC, which is mediated by activation of phosphatidylinositol 3-kinase/Akt and subsequently Nrf2 signaling.     

Variable regulation of glutamate cysteine ligase subunit proteins affects glutathione biosynthesis in response to oxidative stress

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.     

Cellular adaptation mediated through Nrf2-induced glutamate cysteine ligase up-regulation against oxidative stress caused by iron overload in β-thalassemia/HbE patients

Oxidative stress caused as a result of iron overload is implicated in clinical manifestation of beta-thalassemia/haemoglobin E (β-Thal/HbE). In this study, we investigated the cellular adaptation against oxidative stress in β-Thal/HbE patients. Twenty-four paediatric β-Thal/HbE patients and 22 healthy controls were recruited in the study. Blood samples from patients exhibited iron overload, elevation of lipid peroxidation, and marked diminution in the reduced glutathione (GSH) level. However, expression of glutamate-cysteine ligase catalytic (GCLC) subunit, a key enzyme in GSH biosynthesis, was up-regulated when compared with that in controls. GCLC protein levels were correlated with serum iron. There was an enhanced binding activity of the oligonucleotide probe for Nrf2-driven antioxidant response element (ARE) to nuclear protein from blood mononuclear cells of thalassemia subjects. In conclusion, β-Thal/HbE patients exhibit elevated plasma levels of GCLC expression and Nrf2-ARE binding activity, which may account for their adaptive survival response to oxidative stress.     

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.     

Knockdown of Glutamate Cysteine Ligase Catalytic Subunit by siRNA Causes the Gold Nanoparticles-Induced Cytotoxicity in Lung Cancer Cells

Gold nanoparticles (GNPs) have shown promising medical applications in cancer treatment involved in the regulation of intracellular redox balance. Previously, we have reported that GNPs can trigger apoptosis and necrosis in human lung cancer cells (A549) when L-buthionine-sulfoximine (BSO) was used to decrease the expression of intracellular glutathione (GSH). Herein, we investigated the cytotoxicity of GNPs toward lung cancer cells under the glutamate cysteine ligase catalytic subunit (GCLC) was silenced by siRNA. Our results showed that GNPs cause apoptosis and necrosis in cells transfected with GCLC siRNA by elevating intracellular reactive oxygen species (ROS). These findings demonstrated that the regulation of glutathione synthesis by GCLC siRNA in A549 cells can initiate the gold nanoparticles-induced cytotoxicity.     

Multicellular redox regulation in an early-evolving animal treated with glutathione

Redox signaling has emerged as a unifying theme in many seemingly disparate disciplines. Such signaling has been widely studied in bacteria and eukaryotic organelles and is often mediated by reactive oxygen species (ROS). In this context, reduced glutathione (GSH) acts as an important intracellular antioxidant, diminishing ROS and potentially affecting redox signaling. Complementing this cell-level perspective, colonial hydroids can be a useful model for understanding organism-level redox signaling. These simple, early-evolving animals consist of feeding polyps connected by tubelike stolons. Colonies treated exogenously with GSH or reduced glutathione ethyl ester (GEE) were expected to show a morphological change to sheetlike growth typical of low levels of ROS. Contrary to expectations, diminished stolon branching and polyp initiation was observed. Such runnerlike growth is associated with higher levels of ROS, and surprisingly, such higher levels were found in GSH- and GEE-treated colonies. Further investigations show that GSH triggered a feeding response in hydroid polyps, increasing oxygen uptake but at the same time relaxing mitochondrion-rich contractile regions at the base of polyps. Diminished gastrovascular flow and increased emissions of mitochondrial ROS also correlated with the observed runnerlike growth. In contrast to cell-level, "bottom-up" views of redox signaling, here the phenotype may arise from a "top-down" interaction of mitochondrion-rich regions and organism-level physiology. Such multicellular redox regulation may commonly occur in other animals as well.     

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.     

MYC‐driven inhibition of the glutamate‐cysteine ligase promotes glutathione depletion in liver cancer

How MYC reprograms metabolism in primary tumors remains poorly understood. Using integrated gene expression and metabolite profiling, we identify six pathways that are coordinately deregulated in primary MYC‐driven liver tumors: glutathione metabolism; glycine, serine, and threonine metabolism; aminoacyl‐tRNA biosynthesis; cysteine and methionine metabolism; ABC transporters; and mineral absorption. We then focus our attention on glutathione (GSH) and glutathione disulfide (GSSG), as they are markedly decreased in MYC‐driven tumors. We find that fewer glutamine‐derived carbons are incorporated into GSH in tumor tissue relative to non‐tumor tissue. Expression of GCLC, the rate‐limiting enzyme of GSH synthesis, is attenuated by the MYC‐induced microRNA miR‐18a. Inhibition of miR‐18a in vivo leads to increased GCLC protein expression and GSH abundance in tumor tissue. Finally, MYC‐driven liver tumors exhibit increased sensitivity to acute oxidative stress. In summary, MYC‐dependent attenuation of GCLC by miR‐18a contributes to GSH depletion in vivo, and low GSH corresponds with increased sensitivity to oxidative stress in tumors. Our results identify new metabolic pathways deregulated in primary MYC tumors and implicate a role for MYC in regulating a major antioxidant pathway downstream of glutamine.     

The Glutathione System: A New Drug Target in Neuroimmune Disorders

Glutathione (GSH) has a crucial role in cellular signaling and antioxidant defenses either by reacting directly with reactive oxygen or nitrogen species or by acting as an essential cofactor for GSH S-transferases and glutathione peroxidases. GSH acting in concert with its dependent enzymes, known as the glutathione system, is responsible for the detoxification of reactive oxygen and nitrogen species (ROS/RNS) and electrophiles produced by xenobiotics. Adequate levels of GSH are essential for the optimal functioning of the immune system in general and T cell activation and differentiation in particular. GSH is a ubiquitous regulator of the cell cycle per se. GSH also has crucial functions in the brain as an antioxidant, neuromodulator, neurotransmitter, and enabler of neuron survival. Depletion of GSH leads to exacerbation of damage by oxidative and nitrosative stress; hypernitrosylation; increased levels of proinflammatory mediators and inflammatory potential; dysfunctions of intracellular signaling networks, e.g., p53, nuclear factor-κB, and Janus kinases; decreased cell proliferation and DNA synthesis; inactivation of complex I of the electron transport chain; activation of cytochrome c and the apoptotic machinery; blockade of the methionine cycle; and compromised epigenetic regulation of gene expression. As such, GSH depletion has marked consequences for the homeostatic control of the immune system, oxidative and nitrosative stress (O&NS) pathways, regulation of energy production, and mitochondrial survival as well. GSH depletion and concomitant increase in O&NS and mitochondrial dysfunctions play a role in the pathophysiology of diverse neuroimmune disorders, including depression, myalgic encephalomyelitis/chronic fatigue syndrome and Parkinson’s disease, suggesting that depleted GSH is an integral part of these diseases. Therapeutical interventions that aim to increase GSH concentrations in vivo include N-acetyl cysteine; Nrf-2 activation via hyperbaric oxygen therapy; dimethyl fumarate; phytochemicals, including curcumin, resveratrol, and cinnamon; and folate supplementation.     

Glutathione participates in the modulation of starvation-induced autophagy in carcinoma cells

Glutathione (γ-L-glutamyl-L-cysteinyl-glycine, GSH) is the most abundant low molecular weight, thiol-containing compound within the cells and has a primary role in the antioxidant defense and intracellular signaling. Here we demonstrated that nutrient deprivation led to a significant decrease of intracellular GSH levels in three different carcinoma cell lines. This phenomenon was dependent on ABCC1-mediated GSH extrusion, along with GCL inhibition and, to a minor extent, the formation of GSH-protein mixed disulfides that synergistically contributed to the modulation of autophagy by shifting the intracellular redox state toward more oxidizing conditions. Modulation of intracellular GSH by inhibiting its de novo synthesis through incubation with buthionine sulfoximine, or by maintaining its levels through GSH ethyl ester, affected the oxidation of protein thiols, such as PRDXs and consequently the kinetics of autophagy activation. We also demonstrated that thiol-oxidizing or -alkylating agents, such as diamide and diethyl maleate activated autophagy, corroborating the evidence that changes in thiol redox state contributed to the occurrence of autophagy.     

Regulation of protein function by S-glutathiolation in response to oxidative and nitrosative stress.

Protein S-glutathiolation, the reversible covalent addition of glutathione to cysteine residues on target proteins, is emerging as a candidate mechanism by which both changes in the intracellular redox state and the generation of reactive oxygen and nitrogen species may be transduced into a functional response. This review will provide an introduction to the concepts of oxidative and nitrosative stress and outline the molecular mechanisms of protein regulation by oxidative and nitrosative thiol-group modifications. Special attention will be paid to recently published work supporting a role for S-glutathiolation in stress signalling pathways and in the adaptive cellular response to oxidative and nitrosative stress. Finally, novel insights into the molecular mechanisms of S-glutathiolation as well as methodological problems related to the interpretation of the biological relevance of this post-translational protein modification will be discussed.     

Catalase characterization and implication in bleaching of a symbiotic sea anemone

Symbiotic cnidarians are marine invertebrates harboring photosynthesizing microalgae (named zooxanthellae), which produce great amounts of oxygen and free radicals upon illumination. Studying antioxidative balance is then crucial to understanding how symbiotic cnidarians cope with ROS production. In particular, it is suspected that oxidative stress triggers cnidarian bleaching, i.e., the expulsion of zooxanthellae from the animal host, responsible for symbiotic cnidarian mass mortality worldwide. This study therefore investigates catalase antioxidant enzymes and their role in bleaching of the temperate symbiotic sea anemone Anemonia viridis. Using specific separation of animal tissues (ectoderm and endoderm) from the symbionts (zooxanthellae), spectrophotometric assays and native PAGE revealed both tissue-specific and activity pattern distribution of two catalase electrophoretypes, E1 and E2. E1, expressed in all three tissues, presents high sensitivity to the catalase inhibitor aminotriazole (ATZ) and elevated temperatures. The ectodermal E1 form is responsible for 67% of total catalase activity. The E2 form, expressed only within zooxanthellae and their host endodermal cells, displays low sensitivity to ATZ and relative thermostability. We further cloned an ectodermal catalase, which shares 68% identity with mammalian monofunctional catalases. Last, 6 days of exposure of whole sea anemones to ATZ (0.5 mM) led to effective catalase inhibition and initiated symbiont expulsion. This demonstrates the crucial role of this enzyme in cnidarian bleaching, a phenomenon responsible for worldwide climate-change-induced mass mortalities, with catastrophic consequences for marine biodiversity.