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.     

Human glutamate cysteine ligase gene regulation through the electrophile response element

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.     

Mechanisms of glutamate cysteine ligase (GCL) induction by 4-hydroxynonenal

4-Hydroxynonenal (HNE) is one of the major end-products of lipid peroxidation and is increased in response to cellular stress and in many chronic and/or inflammatory diseases. HNE can in turn function as a potent signaling molecule to induce the expression of many genes including glutamate cysteine ligase (GCL), the rate-limiting enzyme in de novo glutathione (GSH) biosynthesis. GSH, the most abundant nonprotein thiol in the cell, plays a key role in antioxidant defense. HNE exposure causes an initial depletion of GSH due to formation of conjugates with GSH, followed by a marked increase in GSH resulting from the induction of GCL. GCL is a heterodimeric protein with a catalytic (or heavy, GCLC) subunit and a modulatory (or light, GCLM) subunit. HNE-mediated induction of both GCL subunits and mRNAs has been reported in rat and human cells in vitro; however, the mechanisms or the signaling pathways mediating the induction of Gclc and Gclm mRNAs by HNE differ between rat and human cells. Activation of the ERK pathway is involved in GCL regulation in rat cells while both the ERK and the JNK pathways appear to be involved in human cells. Downstream, MAPK activation leads to increased AP-1 binding, which mediates GCL induction. Some studies suggest a role for the EpRE element as well. As the concentrations of HNE used in all of the studies reviewed are comparable to what may be found in vivo, this makes the findings summarized in this review potentially relevant to GCL regulation in human health and disease.     

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.     

Induction of lung glutathione and glutamylcysteine ligase by 1,4-phenylenebis(methylene)selenocyanate and its glutathione conjugate: role of nuclear factor-erythroid 2-related factor 2

The synthetic organoselenium agent 1,4-phenylenebis(methylene)selenocyanate (p-XSC) and its glutathione (GSH) conjugate (p-XSeSG) are potent chemopreventive agents in several preclinical models. p-XSC is also an effective inducer of GSH in mouse lung. Our objectives were to test the hypothesis that GSH induction by p-XSC occurs through upregulation of the rate-limiting GSH biosynthetic enzyme glutamylcysteine ligase (GCL), through activation of antioxidant response elements (AREs) in GCL genes via activation of nuclear factor-erythroid 2-related factor 2 (Nrf2). p-XSC feeding (10 ppm Se) increased GSH (230%) and upregulated the catalytic subunit of GCL (GCLc) (55%), extracellular-related kinase (220%), and nuclear Nrf2 (610%) in lung but not liver after 14 days in the rat (P<0.05). Similarly, p-XSeSG feeding (10 ppm) induced lung GCLc (88%) and GSH (200%) (P<0.05), whereas the naturally occurring selenomethionine had no effect. Both p-XSC and p-XSeSG activated a luciferase reporter in HepG2 ARE-reporter cells up to threefold for p-XSC and greater than or equal to fivefold for p-XSeSG. Luciferase activation by p-XSeSG was associated with enhanced levels of GSH, GCLc, and nuclear Nrf2, which were significantly reduced by co-incubation with short interfering RNA targeting Nrf2. The dependence of GCL induction on Nrf2 was confirmed in Nrf2-deficient mouse embryonic fibroblasts, in which p-XSeSG induced GCL subunits in wild-type but not in Nrf2-deficient cells (P<0.05). These results indicate that p-XSC may act through the Nrf2 pathway in vivo and that p-XSeSG is the putative metabolite responsible for such activation, thus offering p-XSeSG as a less toxic, yet highly efficacious, inducer of GSH.     

Glutamate cysteine ligase catalysis: dependence on ATP and modifier subunit for regulation of tissue glutathione levels

Glutamate cysteine ligase (GCL), which synthesizes gamma-glutamyl-cysteine (gamma-GC), is the rate-limiting enzyme in GSH biosynthesis. gamma-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 gamma-GC synthesis by GCLholo. GCLM decreased the Km for ATP by approximately 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 gamma-GC synthesis; this difference in catalytic efficiency of GCLholo versus GCLC allowed us to derive a mathematical relationship for gamma-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 gamma-GC synthesis. On the other hand, our results from kidney suggest that gamma-GC synthesis may be controlled post-translationally.     

Pharmacological rescue of the 14CoS/14CoS mouse: hepatocyte apoptosis is likely caused by endogenous oxidative stress

Whereas ch/ch wild-type mice and ch/14CoS heterozygotes are viable, 14CoS/14CoS mice homozygous for a 3800 kb deletion on chromosome 7 die during the first day postpartum. Death is caused by disruption of the fumarylacetoacetate hydrolase (Fah) gene; absence of FAH, final enzyme in the tyrosine catabolism pathway, leads to accumulation of reactive electrophilic intermediates. In this study, we kept 14CoS/14CoS mice alive for 60 d with oral 2-(2-nitro-4-trifluoromethyl-benzyol)-1,3-cyclohexanedione (NTBC), an inhibitor of p-hydroxyphenylpyruvate dioxygenase, second enzyme in the tyrosine catabolic pathway. The 70% of NTBC-treated 14CoS/14CoS mice that survived 60 d showed poor growth and developed corneal opacities, compared with ch/14CoS littermates; NTBC-rescued Fah(-/-) knockout mice did not show growth retardation or ocular toxicity. NTBC-rescued 14CoS/14CoS mice also exhibited a striking oxidative stress response in liver and kidney, as measured by lower GSH levels and mRNA induction of four genes: glutamate cysteine ligase catalytic (Gclc) and modifier (Gclm) subunits, NAD(P)H:quinone oxidoreductase (Nqo1), and heme oxygenase-1 (Hmox1). Withdrawal of NTBC for 24-48 h from rescued adult 14CoS/14CoS mice resulted in severe apoptosis of the liver, detected histologically and by cytochrome c release from the mitochondria, increased caspase 3-like activity, and further decreases in GSH content. In kidney, proximal tubular epithelial cells were abnormal. Human hereditary tyrosinemia type I (HT1), caused by mutations in the FAH gene, is an autosomal recessive disorder in which the patient usually dies of liver fibrosis and cirrhosis during early childhood; NTBC treatment is known to prolong HT1 children's lives-although liver fibrosis, cirrhosis, hepatocarcinoma, and corneal opacities sometimes occur. The mouse data in the present study are consistent with the possibility that endogenous oxidative stress-induced apoptosis may be the underlying cause of liver pathology seen in NTBC-treated HT1 patients.     

Over expression of glutamate cysteine ligase increases cellular resistance to H2O2-induced DNA single-strand breaks.

Hydrogen peroxide (H2O2) can cause single strand DNA breaks (ssDNA) in cells when the mechanisms normally in place to reduce it are overwhelmed. Such mechanisms include catalase, glutathione peroxidases (GPx), and peroxiredoxins. The relative importance of these enzymes in H2O2 reduction varies with cell and tissue type. The role of the GPx cofactor glutathione (GSH) in oxidative defense can be further understood by modulating its synthesis. The first and rate-limiting enzyme in GSH synthesis is glutamate-cysteine ligase (GCL), which has a catalytic subunit (Gclc) and a modifier subunit (Gclm). Using mouse hepatoma cells we evaluated the effects of GCL over expression on H2O2-induced changes in GSH and ssDNA break formation with the single cell gel electrophoresis assay (SCG or comet assay), and the acridine orange DNA unwinding flow cytometry assay (AO unwinding assay). Cells over expressing GCL had higher GSH content than control cells, and both SCG and AO unwinding assays revealed that cells over expressing GCL were significantly more resistant to H2O2-induced ssDNA break formation. Furthermore, using the AO unwinding assay, the prevalence of H2O2-induced breaks in different phases of the cell cycle was not different, and the degree of protection afforded by GCL over expression was also not cell cycle phase dependent. Our results support the hypothesis that GCL over expression enhanced GSH biosynthesis and protected cells from H2O2-induced DNA breaks. These results also suggest that genetic polymorphisms that affect GCL expression may be important determinants of oxidative DNA damage and cancer.     

Co-ordinate expression of cytochrome c oxidase subunit III and VIc mRNAs in rat tissues.

Cytochrome c oxidase (EC 1.9.3.1) is an enzyme which is composed of subunits derived from both the mitochondrial and the nuclear genomes. To determine whether or not the expression of these two genomes is co-ordinated at the mRNA level, we have examined the steady-state levels of mRNAs coding for cytochrome c oxidase subunit III (mitochondrially encoded) and subunit VIc (nuclear-encoded) in rat tissues. This was compared with the tissue concentration of the holoenzyme, which was estimated by measuring cytochrome c oxidase enzyme activity. The tissues (heart, brain, liver, kidney, soleus muscle and superficial white vastus muscle) possessed a 13-fold range of enzyme activity, which was highest in heart and lowest in the superficial vastus muscle. Specific subunit mRNA levels were quantified by using slot-blot hybridization of cDNA probes to total tissue RNA. The highest values for subunit III and Vlc mRNA tissue contents were found in kidney, followed by liver and heart (40-60% of that of kidney). The white vastus muscle contained the lowest subunit mRNA level (15% of that of kidney). Although some variability was apparent within each tissue, a parallel pattern of mRNA expression of the nuclear- and mitochondrially encoded subunits was observed. Differences between muscle (heart, vastus and soleus) and non-muscle tissues were noted in the relationship between mRNA and protein levels of expression. Thus, although this suggests that tissue-specific regulatory processes operate, the steady-state expression of subunit III and subunit Vlc mRNAs appears to be co-ordinately regulated.     

The Catalytic Subunit of Drosophila Glutamate-Cysteine Ligase Is a Nucleocytoplasmic Shuttling Protein

GSH concentration is considerably lower in the nucleus than in the cytoplasm; however, it is significantly elevated during active cell proliferation. The main purpose of this study was to understand the mechanism underlying these variations in nuclear/cytoplasmic distribution of GSH. The rate-limiting step in the de novo GSH biosynthesis pathway is catalyzed by glutamate cysteine ligase (GCL), a heterodimer, composed of a catalytic subunit (GCLc) and a modulatory subunit (GCLm). In Drosophila, GCLc, but not GCLm, contains a nuclear localization signal (NLS). Drosophila S2 cells, constitutively expressing regular GCLc protein or expressing GCLc protein with a mutated NLS motif, were generated by transfection. In quiescent S2 cells, GCLc is aggregated in the perinuclear cytosol and the nucleus, whereas GLCm resides solely in the cytosol. In actively proliferating S2 cells, expressing the normal NLS motif, GCLc migrates from the perinuclear cytoplasm into the nucleus, and the nuclear GSH level becomes elevated; in contrast, in proliferating cells, expressing the mutated NLS motif, neither does the GCLc migrate into the nucleus nor does the nuclear GSH amount rise. In S2 cells expressing wild type GCLc, perturbation of cellular redox state by exposure to cadmium resulted in the migration of GCLc into the nucleus but not in cells expressing GCLc with the mutated NLS motif. Overall, results indicated that GSH biosynthesis in the nucleus is associated with migration of only the GCLc subunit from the cytoplasm into the nucleus, and this migration requires the presence of an intact NLS.The tripeptide, γ-glutamylcysteinylglycine or GSH, is the most abundant intracellular nonprotein thiol. It serves multiple physiological functions, including maintenance of redox homeostasis, providing reducing equivalents for the elimination of reactive oxygen species, protection against electrophilic xenobiotics, maintenance of protein structure, and storage/transport of l-cysteine. GSH is synthesized de novo by two ATP-dependent consecutive reactions: ligation of l-glutamate to l-cysteine by the activity of glutamate-cysteine ligase (GCL²; EC 6.3.2.2), a rate-limiting step in the pathway, followed by the coupling of glycine to γ-glutamylcysteine by glutathione synthase (EC 6.3.2.3). The GCL holoenzyme is heterodimeric, consisting of a catalytic (GCLc) and a modifier (GCLm) subunit, each encoded by a unique gene. The intracellular ratios of GLCc and GCLm are, however, not necessarily equimolar and may be altered under conditions of oxidative stress. Although GCLc by itself is fully competent to catalyze the biosynthesis of GSH (1), dimerization with GCLm lowers the K_m for glutamate and also decreases sensitivity to feedback inhibition by GSH (2).Overexpression of GCL subunits in cultured cells has been reported to increase GSH production and confer enhanced protection against oxidative stress (3), apoptosis (4), and oxidant-induced DNA lesions (5). We have shown that overexpressions of GCLc or GCLm boost GSH biosynthesis and extend life span in Drosophila melanogaster (6). In contrast, inhibition of GCL activity results in decreased GSH levels, enhanced susceptibility to oxidative or nitrosative stress, increased DNA damage, and cell cycle arrest (7, 8). The homozygous knock-out for the catalytic GCLc subunit is embryonic lethal (9).GSH is predominantly synthesized in the cytoplasm, but its levels greatly vary among the different intracellular compartments, such as the nucleus, mitochondria, endoplasmic reticulum, and cytosol (10–12). Currently available evidence suggests the existence of two alternate mechanisms for the differential distribution of GSH in subcellular compartments: (i) GSH may first be synthesized in the cytoplasm and then transported into the organelles either actively or via passive diffusion; (ii) GSH may be synthesized in those organelles that display activities of GCL and glutathione synthase (13, 14).It has been demonstrated that GSH levels in the nucleus of proliferating cells are much higher than those in the confluent cells (15); however, the mechanism underlying this variation is presently unclear. In the present study, we provide evidence that GSH is synthesized in the nucleus and that this synthesis is dependent upon the shuttling of the GCLc, but not the GCLm, subunit from the cytoplasm to the nucleus. The ability of GCLc to migrate into the nucleus is due to the presence of a nuclear localization signal (NLS).     

PP2A-A/在金鱼及斑马鱼发育中的分化表达模式

以金鱼和斑马鱼为研究对象,运用RT-PCR和Western Blot技术分析蛋白磷酸酶2A(PP2A)结构亚基A(PP2A-A/)在金鱼、斑马鱼成体9种组织和12个发育时期胚胎中mRNA和蛋白水平的表达情况,得到其分化表达模式为:(1)在mRNA水平上,PP2A-A/在金鱼、斑马鱼9种组织中具有较强表达;种属差异性和组织差异性均较大;结构亚基A的两亚型A和A的表达存在差异。(2)在蛋白水平上,PP2A-A/在金鱼、斑马鱼9种组织中均有表达;种属差异性不大但出现明显的组织差异性。(3)PP2A-A/mRNA在金鱼和斑马鱼卵裂期到囊胚期胚胎中大量存在,PP2A-AmRNA在金鱼眼色素期量剧增推测其对金鱼眼色素的形成至关重要。(4)PP2A-A/基因在金鱼、斑马鱼12个发育时期胚胎中均有较高水平的蛋白存在,提示其为维持胚胎的正常发育发挥重要作用。       

Differential tissue expression of the glutathione transferase multigene family.

The content of GSH transferase mRNAs in poly(A)-containing RNA isolated from eight rat tissues was examined by immunoprecipitation of cell-free translation products and by Northern blotting. Considerable tissue-specific distribution and heterogeneity of immunoprecipitable GSH transferase subunits 1 and 2 synthesized in vitro was observed. These results were confirmed by Northern blotting using a 32P-labelled subunit 1 cDNA probe. The same probe, used in a Southern blot analysis of genomic DNA, provided confirmation that GSH transferase subunits 1 and 2 comprise a multigenic family in the rat. The results show that the selection of cDNA clones coding for chosen subunits can be made easier by making use of qualitative and quantitative tissue differences in GSH transferase mRNAs.     

A universal oligonucleotide probe for acetylcholine receptor genes. Selection and sequencing of cDNA clones for the mouse muscle beta subunit

A region of 25 nucleotides is highly conserved in genes coding for the alpha, beta, gamma, and delta subunits of the nicotinic acetylcholine receptor (AChR) of human, mouse, calf, chicken, and Torpedo. Based on this observation, a 2-fold degenerate oligonucleotide was synthesized and used as a probe to screen a cDNA library made from a mouse myogenic cell line. Clones coding for the beta, gamma, and delta subunits were identified by the probe. The protein sequence deduced from the beta subunit clones codes for a precursor polypeptide of 501 amino acids with a calculated molecular weight of 56,930 daltons, which includes a signal peptide of 23 amino acids. The protein sequence and structural features of the beta subunits of mouse, calf, and Torpedo are conserved. A clone coding for the mouse gamma subunit was isolated, and its identity was confirmed by alignment of its sequence to previously published cDNA sequences for the mouse and calf gamma subunits. The clone contained approximately 200 nucleotides more at its 3' end untranslated region than a mouse gamma clone recently described. Northern blot analysis, utilizing as probes these beta and gamma subunit cDNAs and previously characterized alpha and delta subunit cDNAs, shows that the steady-state levels of the four AChR mRNAs increase coordinately during terminal differentiation of cultured C2 and C2i mouse myoblasts. The increase in mRNA levels can account for the rise of cell surface receptors during myogenesis and suggests that the muscle AChR genes may be regulated during development by a common mechanism. Utilization of this oligonucleotide probe should prove useful for screening a variety of libraries made from different species and tissues which are known to express AChRs.