Inducible expression of the gamma-glutamylcysteine synthetase light subunit by t-butylhydroquinone in HepG2 cells is not dependent on an antioxidant-responsive element.
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ABSTRACT:
SUBMITTER: Galloway DC
PROVIDER: S-EPMC1219901 | biostudies-other | 1998 Dec
REPOSITORIES: biostudies-other
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Project description:gamma-Glutamylcysteine synthetase (GCS) is the rate-limiting enzyme in the biosynthesis of glutathione and is composed of a heavy and a light subunit. Although the heavy subunit is enzymically active alone, the light subunit plays an important regulatory role by making the holoenzyme function more efficiently. In the current study we examined whether conditions which are known to influence gene expression of the heavy subunit also influence that of the light subunit, and the mechanisms involved. Treatment of cultured rat hepatocytes with hormones such as insulin and hydrocortisone, or plating hepatocytes under low cell density increased the steady-state mRNA level of the heavy subunit only. Treatment with diethyl maleate (DEM), buthionine sulphoximine (BSO) and t-butylhydroquinone (TBH) increased the steady state mRNA level and gene transcription rates of both subunits. These treatments share in common their ability to induce oxidative stress and activate nuclear factor kappa B (NF-kappa B). Treatment with protease inhibitors 7-amino-1-chloro-3-tosylamido-2-heptanone (TLCK) or L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK) had no influence on the basal NF-kappa B and GCS subunit mRNA levels, but blocked the activation of NF-kappa B by DEM, BSO and TBH, and the increase in GCS heavy subunit mRNA level by BSO and TBH. On the other hand, the DEM-, BSO- and TBH-induced increase in GCS light-subunit mRNA level was unaffected by TLCK and TPCK. Thus only the heavy subunit is hormonally regulated and growth sensitive, whereas both subunits are regulated by oxidative stress. Signalling through NF-kappa B is involved only in the oxidative-stress-mediated changes in the heavy subunit gene expression.
| S-EPMC1218650 | biostudies-other
Project description:gamma-Glutamylcysteine synthetase (GCS), the rate-limiting enzyme in the de novo synthesis of GSH, is a heterodimer, consisting of a catalytic (GCSh) and a regulatory subunit (GCSl). We previously demonstrated that the constitutive and beta-naphthoflavone (beta-NF)-induced expression of the GCSh gene is mediated by a distal antioxidant response element (ARE), ARE4, located 3.1 kb upstream of the transcriptional start site [Mulcahy, Wartman, Bailey and Gipp (1997) J. Biol. Chem. 272, 7445-7454]. ARE4 consists of a consensus ARE sequence (5'-GTGACTCAGCG-3') containing an embedded PMA-responsive element (TRE, underlined). The relative significance of the two overlapping response elements to constitutive and beta-NF-induced expression of the GCSh gene was determined by mutational analyses. The internal activator protein-1 (AP-1)-binding sequence mediated constitutive expression of promoter/reporter transgenes, but was not required for beta-NF responsiveness. In gel-shift experiments, the TRE was necessary for binding of proteins from nuclear extracts prepared from untreated HepG2 cells. In contrast, induction by beta-NF was dependent on an intact ARE sequence, particularly the terminal GC box of ARE4. The GC box of ARE4 was shown to be essential for both basal and beta-NF-induced expression of reporter constructs. This element also influenced binding of nuclear proteins to ARE4, specifically in extracts isolated from beta-NF-treated HepG2 cells. Because previous studies indicated that ARE4 may co-operate with a separate putative ARE, the role of the neighbouring sequence (ARE3), located 34 bases downstream of ARE4, was also evaluated. Mutation of this element within a GCSh promoter/reporter did not modify the basal or beta-NF-induced expression of the transgene, demonstrating that ARE3 does not influence the constitutive or beta-NF-induced expression of the GCSh gene.
| S-EPMC1219492 | biostudies-other
Project description:gamma-Glutamylcysteine synthetase (GCS) is reported to catalyse the rate-limiting step in glutathione biosynthesis, and is a heterodimer composed of a catalytic subunit [heavy subunit (GCSh) of Mr 73000] and a regulatory subunit [light subunit (GCSl) of Mr 31000]. In the present study, we have demonstrated for the first time a potential role for GCSl in resistance towards doxorubicin and cadmium chloride. Addition of recombinant GCSl to HeLa cell extracts in vitro was found to result in an increase in GCS activity of between 2- and 3-fold. Transient transfections of COS-1 cells with the GCSl cDNA cause an increase in GCS activity of approx. 2-fold, and a small but significant (P=0.008) increase in glutathione levels from 126.9+/-4. 2 nmol/mg protein to 178.8+/-19.1 nmol/mg protein. We proceeded to make a HeLa cell line (LN73), which stably overexpresses GCSl. These cells overexpress GCSl approx. 20-fold above basal levels. LN73 was found to have a 2-fold increase in GCS activity (437.3+/-85.2 pmol/min per mg) relative to the control cell line, HL9 (213.4+/-71. 8 pmol/min per mg). In contrast with the transient transfections in COS-1 cells, stable overexpression of GCSl was found not to be associated with an increase in glutathione content. However, when the LN73 and HL9 cells were treated with the glutathione-depleting agent, diethylmaleate, the LN73 cells were found to have an enhanced ability to regenerate glutathione, compared with HL9 cells. The cell lines were treated with various anti-cancer drugs, and their cytotoxicity was examined. No obvious differences in toxicity were observed between the different cell lines following treatment with cisplatin and melphalan. The redox-cycling agent doxorubicin, however, was found to be more toxic (approx. 2-fold) to the HL9 cells than the LN73 cells. When the cells were treated with the carcinogenic transition-metal compound, cadmium chloride, LN73 cells were found to be approx. 3-fold more resistant than HL9 cells.
| S-EPMC1220010 | biostudies-other
Project description:?-glutamylcysteine synthetase (Gcs) is a vital enzyme catalyzing the first and rate limiting step in the trypanothione biosynthesis pathway, the ATP-dependent ligation of L-Glutamate and L-Cysteine to form gamma-glutamylcysteine. The Trypanothione biosynthesis pathway is unique metabolic pathway essential for trypanosomatid survival rendering Gcs as a potential drug target. Here we report the cloning, expression, purification and characterization of L. donovani Gcs. Three other constructs of Gcs (GcsN, GcsC and GcsT) were designed on the basis of S. cerevisiae and E. coli Gcs crystal structures. The study shows Gcs possesses ATPase activity even in the absence of substrates L-glutamate and L-Cysteine. Divalent ions however plays an indispensable role in LdGcs ATPase activity. Isothermal titration calorimetry and fluorescence studies illustrates that L. donovani Gcs binds substrate in order ATP >L-glutamate>L-cysteine with Glu 92 and Arg 498 involved in ATP hydrolysis and Glu 92, Glu 55 and Arg 498 involved in glutamate binding. Homology modeling and molecular dynamic simulation studies provided the structural rationale of LdGcs catalytic activity and emphasized on the possibility of involvement of three Mg2+ ions along with Glutamates 52, 55, 92, 99, Met 322, Gln 328, Tyr 397, Lys 483, Arg 494 and Arg 498 in the catalytic function of L. donovani Gcs.
| S-EPMC5613772 | biostudies-literature
Project description:Glutathione (GSH) is a key component of plant antioxidant defenses. We have sought to determine how the rate-limiting step in GSH biosynthesis, catalyzed by gamma-glutamylcysteine synthase (gammaECS) is regulated in Arabidopsis. Functional complementation of a yeast mutant deficient in this enzyme with an Arabidopsis expression library yielded two cDNAs with sequence identical to the previously described AtgammaECS. Nevertheless, the cellular concentration of GSH in these transformants was only 10% of wild-type concentrations and this was not a result of Cys availability. To explore the possibility that Arabidopsis gammaECS requires additional factors for full catalytic activity, we analyzed the GSH levels and the enzyme activities and transcript levels of both enzymes of the GSH biosynthetic pathway in Arabidopsis suspension cultures subjected to a variety of stresses that raise GSH levels. Our results demonstrate rapid posttranscriptional activation of Arabidopsis gammaECS. The implications of these findings for the mechanisms by which GSH concentrations are regulated during plant-stress responses are discussed.
| S-EPMC21763 | biostudies-literature
Project description:We have shown that in HepG2 cells treatment with 75 microM t-butylhydroquinone (tBHQ) results in a 2.5-fold increase in glutathione concentration, as part of an adaptive response to chemical stress. In these cells the elevation in intracellular glutathione level was found to be accompanied by an increase of between 2-fold and 3-fold in the level of the 73 kDa catalytic subunit of gamma-glutamylcysteine synthetase (heavy subunit, GCSh) and the 31 kDa regulatory subunit (light subunit, GCSl). Levels of GCSh and GCSl mRNA were increased by up to 5-fold in HepG2 cells in response to tBHQ. To study the transcriptional regulation of GCSl, we subcloned 6.7 kb of the upstream region of the human GCSl gene (GLCLR) from a genomic clone isolated from a P1 lymphoblastoid cell line genomic library. HepG2 cells were transfected with GLCLR promoter reporter constructs and treated with tBHQ. This resulted in an induction of between 1.5-fold and 3.5-fold in reporter activity, indicating that transcriptional regulation of GLCLR is likely to contribute to the induction of GCSl by tBHQ in HepG2 cells. Sequence analysis of the promoter region demonstrated the presence of putative enhancer elements including AP-1 sites and an antioxidant-responsive element, which might be involved in the observed induction of the GLCLR promoter.
| S-EPMC1218892 | biostudies-other
Project description:Trypanothione (T(SH)2) is the main antioxidant metabolite for peroxide reduction in Trypanosoma cruzi; therefore, its metabolism has attracted attention for therapeutic intervention against Chagas disease. To validate drug targets within the T(SH)2 metabolism, the strategies and methods of Metabolic Control Analysis and kinetic modeling of the metabolic pathway were used here, to identify the steps that mainly control the pathway fluxes and which could be appropriate sites for therapeutic intervention. For that purpose, gamma-glutamylcysteine synthetase (γECS), trypanothione synthetase (TryS), trypanothione reductase (TryR) and the tryparedoxin cytosolic isoform 1 (TXN1) were separately overexpressed to different levels in T. cruzi epimastigotes and their degrees of control on the pathway flux as well as their effect on drug resistance and infectivity determined. Both experimental in vivo as well as in silico analyses indicated that γECS and TryS control T(SH)2 synthesis by 60-74% and 15-31%, respectively. γECS overexpression prompted up to a 3.5-fold increase in T(SH)2 concentration, whereas TryS overexpression did not render an increase in T(SH)2 levels as a consequence of high T(SH)2 degradation. The peroxide reduction flux was controlled for 64-73% by TXN1, 17-20% by TXNPx and 11-16% by TryR. TXN1 and TryR overexpression increased H2O2 resistance, whereas TXN1 overexpression increased resistance to the benznidazole plus buthionine sulfoximine combination. γECS overexpression led to an increase in infectivity capacity whereas that of TXN increased trypomastigote bursting. The present data suggested that inhibition of high controlling enzymes such as γECS and TXN1 in the T(SH)2 antioxidant pathway may compromise the parasite's viability and infectivity.
| S-EPMC6581782 | biostudies-literature
Project description:We previously reported that the activity of gamma-glutamylcysteine synthetase (GCS; EC 6.3.2.2), the rate-limiting enzyme in GSH synthesis, can be acutely inhibited approximately 20-40% by agonists of various signal transduction pathways in rat hepatocytes [Lu, Kuhlenkamp, Garcia-Ruiz and Kaplowitz (1991) J. Clin. Invest. 88, 260-269]. We have now examined the possibility that GCS is phosphorylated directly by activation of protein kinase A (PKA), protein kinase C (PKC) and Ca2+/calmodulin-dependent kinase II (CMK). Phosphorylation of GCS was studied using both purified rat kidney GCS and cultured rat hepatocytes by immunoprecipitating the reaction product with specific rabbit anti-(rat GCS heavy subunit) (anti-GCS-HS) antibodies. All three kinases, PKA, PKC and CMK, phosphorylated rat kidney GCS-HS in a Mg(2+)-concentration-dependent manner, with the highest degree of phosphorylation occurring at 20 mM Mg2+. The maximum incorporation of phosphate in mol/mol of GCS was 1.17 for PKA, 0.70 for PKC and 0.62 for CMK. The degree of phosphorylation was correlated with the degree of loss of GCS activity, and no additional inhibition occurred when GCS was phosphorylated by all three kinases, suggesting that the kinases phosphorylated the same site(s). Phosphoamino analysis showed that all three kinases phosphorylated serine and threonine residues. Two-dimensional phosphopeptide mapping demonstrated that all three kinases phosphorylated the same five peptides, both PKA and PKC phosphorylated two other peptides, and only PKA phosphorylated one additional peptide. Phosphorylation of GCS decreased its Vmax for cysteine and glutamate without changing its K(m). Finally, treatment of cultured rat hepatocytes with dibutyryl cAMP and phenylephrine significantly increased the phosphorylation of GCS, suggesting a potentially important physiological role. In summary, we have demonstrated that GCS is phosphorylated and suggest that phosphorylation/dephosphorylation may regulate GCS activity.
| S-EPMC1217934 | biostudies-other
Project description:BackgroundGamma-glutamylcysteine synthetase (γ-ECS) is a rate-limiting enzyme in glutathione biosynthesis and plays a key role in plant stress responses. In this study, the endogenous expression of the Caragana korshinskii γ-ECS (Ckγ-ECS) gene was induced by PEG 6000-mediated drought stress in the leaves of C. korshinskii. and the Ckγ-ECS overexpressing transgenic Arabidopsis thaliana plants was constructed using the C. korshinskii. isolated γ-ECS.ResultsCompared with the wildtype, the Ckγ-ECS overexpressing plants enhanced the γ-ECS activity, reduced the stomatal density and aperture sizes; they also had higher relative water content, lower water loss, and lower malondialdehyde content. At the same time, the mRNA expression of stomatal development-related gene EPF1 was increased and FAMA and STOMAGEN were decreased. Besides, the expression of auxin-relative signaling genes AXR3 and ARF5 were upregulated.ConclusionsThese changes suggest that transgenic Arabidopsis improved drought tolerance, and Ckγ-ECS may act as a negative regulator in stomatal development by regulating the mRNA expression of EPF1 and STOMAGEN through auxin signaling.
| S-EPMC8485494 | biostudies-literature
Project description:Rubber tree is a major commercial source of natural rubber. Latex coagulation is delayed by thiols, which belong to the important type of antioxidants in laticifer submembrane, and is composed of glutathione (GSH), cysteine, and methionine. The rate-limiting enzyme, ?-ECS, plays an important role in regulating the biosynthesis of glutathione under any environment conditions. To understand the relation between ?-ECS and thiols and to correlate latex flow with one-time tapping and continuous tapping, we cloned and derived the full length of one ?-ECS from rubber tree latex (Hb?-ECS1). According to qPCR analysis, the expression levels of Hb?-ECS1 were induced by tapping and Ethrel stimulation, and the expression was related to thiols content in the latex. Continuous tapping induced injury, and the expression of Hb?ECS1 increased with routine tapping and Ethrel-stimulation tapping (more intensive tapping). According to expression in long-term flowing latex, the gene was related to the duration of latex flow. Hb?ECS1 was expressed in E. coli Rosetta using pET-sumo as an expression vector and the recombinant enzyme was purified; then we achieved 0.827?U/mg specific activity and about 66 kDa molecular weight. The present study can help us understand the complex role of Hb?-ECS in thiols biosynthesis, which is influenced by tapping.
| S-EPMC4935901 | biostudies-literature