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Glutamylcysteine Synthetase (glutamylcysteine + synthetase)
Selected AbstractsCrystallization and preliminary crystallographic analysis of bifunctional ,-glutamylcysteine synthetase,glutatione synthetase from Streptococcus agalactiaeACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2009Yasunori Nakashima ,-Glutamylcysteine synthetase,glutathione synthetase (,GCS-GS) is a bifunctional enzyme that catalyzes two consecutive steps of ATP-dependent peptide formation in glutathione biosynthesis. Streptococcus agalactiae,GCS-GS is a target for the development of potential therapeutic agents. ,GCS-GS was crystallized using the sitting-drop vapour-diffusion method. The crystals grew to dimensions of 0.3 × 0.2 × 0.2,mm under reducing conditions with 5,mM TCEP. X-ray data were collected to 2.8,Å resolution from a tetragonal crystal that belonged to space group I41. [source] Cadmium induced oxidative stress influence on glutathione metabolic genes of Camellia sinensis (L.) O. KuntzeENVIRONMENTAL TOXICOLOGY, Issue 4 2007Prashant Mohanpuria Abstract Glutathione, a tripeptide with sulfhydryl (-SH) group is a very crucial compound primarily involved in redox balance maintenance of the cellular environment. In this study, we monitored the influence of Cd exposure on the transcript levels of glutathione metabolic genes in bud tissues, the youngest leaf, of Camellia sinensis L. In addition, some physiochemical parameters were also studied. Cd exposure decreased chlorophyll and protein contents, while increase was observed in lipid peroxidation upon Cd treatments. These changes were found to be concentration and duration dependent, indicating the occurrence of oxidative stress upon Cd exposure. The transcript levels of glutathione biosynthetic genes viz. ,-glutamylcysteine synthetase (,-ECS) and glutathione synthetase (GSHS) increased upon Cd exposure. Furthermore, transcript levels of glutathione reductase (GR), an enzyme involved in reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH), also showed upregulation on Cd exposure. However, the transcript levels of glutathione-S-transferase (GST), an enzyme involved in forming metal,GSH complex and help in sequestration of high levels of metal ions to vacuole, did not show any change on Cd treatment. This study document that Cd exposure induces oxidative stress in Camellia sinensis and the upregulation in transcript levels of glutathione metabolic genes except GST have suggested the role of these enzymes in the protection of plants from high level Cd exposure. © 2007 Wiley Periodicals, Inc. Environ Toxicol 22: 368,374, 2007. [source] Sulfur assimilation and glutathione metabolism under cadmium stress in yeast, protists and plantsFEMS MICROBIOLOGY REVIEWS, Issue 4 2005David Mendoza-Cózatl Abstract Glutathione (,-glu-cys-gly; GSH) is usually present at high concentrations in most living cells, being the major reservoir of non-protein reduced sulfur. Because of its unique redox and nucleophilic properties, GSH serves in bio-reductive reactions as an important line of defense against reactive oxygen species, xenobiotics and heavy metals. GSH is synthesized from its constituent amino acids by two ATP-dependent reactions catalyzed by ,-glutamylcysteine synthetase and glutathione synthetase. In yeast, these enzymes are found in the cytosol, whereas in plants they are located in the cytosol and chloroplast. In protists, their location is not well established. In turn, the sulfur assimilation pathway, which leads to cysteine biosynthesis, involves high and low affinity sulfate transporters, and the enzymes ATP sulfurylase, APS kinase, PAPS reductase or APS reductase, sulfite reductase, serine acetyl transferase, O -acetylserine/O -acetylhomoserine sulfhydrylase and, in some organisms, also cystathionine ,-synthase and cystathionine ,-lyase. The biochemical and genetic regulation of these pathways is affected by oxidative stress, sulfur deficiency and heavy metal exposure. Cells cope with heavy metal stress using different mechanisms, such as complexation and compartmentation. One of these mechanisms in some yeast, plants and protists is the enhanced synthesis of the heavy metal-chelating molecules GSH and phytochelatins, which are formed from GSH by phytochelatin synthase (PCS) in a heavy metal-dependent reaction; Cd2+ is the most potent activator of PCS. In this work, we review the biochemical and genetic mechanisms involved in the regulation of sulfate assimilation-reduction and GSH metabolism when yeast, plants and protists are challenged by Cd2+. [source] GSH2, a gene encoding ,-glutamylcysteine synthetase in the methylotrophic yeast Hansenula polymorphaFEMS YEAST RESEARCH, Issue 3 2002Vira M Ubiyvovk Abstract The GSH2 gene, encoding Hansenula polymorpha,-glutamylcysteine synthetase, was cloned by functional complementation of a glutathione (GSH)-deficient gsh2 mutant of H. polymorpha. The gene was isolated as a 4.3-kb XbaI fragment that was capable of restoring GSH synthesis, heavy-metal resistance and cell proliferation when introduced into gsh2 mutant cells. It possesses 53% identical and 69% similar amino acids compared with the Candida albicans homologue (Gcs1p). In comparison to the Saccharomyces cerevisiae homologue (Gsh1p), it possesses 47% identical and 61% similar amino acids. The GSH2 sequence appears in the GenBank database under accession No. AF435121. [source] Necrostatin-1 protects against glutamate-induced glutathione depletion and caspase-independent cell death in HT-22 cellsJOURNAL OF NEUROCHEMISTRY, Issue 5 2007Xingshun Xu Abstract Glutamate, a major excitatory neurotransmitter in the CNS, plays a critical role in neurological disorders such as stroke and Parkinson's disease. Recent studies have suggested that glutamate excess can result in a form of cell death called glutamate-induced oxytosis. In this study, we explore the protective effects of necrostatin-1 (Nec-1), an inhibitor of necroptosis, on glutamate-induced oxytosis. We show that Nec-1 inhibits glutamate-induced oxytosis in HT-22 cells through a mechanism that involves an increase in cellular glutathione (GSH) levels as well as a reduction in reactive oxygen species production. However, Nec-1 had no protective effect on free radical-induced cell death caused by hydrogen peroxide or menadione, which suggests that Nec-1 has no antioxidant effects. Interestingly, the protective effect of Nec-1 was still observed when cellular GSH was depleted by buthionine sulfoximine, a specific and irreversible inhibitor of glutamylcysteine synthetase. Our study further demonstrates that Nec-1 significantly blocks the nuclear translocation of apoptosis-inducing factor (a marker of caspase-independent programmed cell death) and inhibits the integration of Bcl-2/adenovirus E1B 19 kDa-interacting protein 3 (a pro-death member of the Bcl-2 family) into the mitochondrial membrane. Taken together, these results demonstrate for the first time that Nec-1 prevents glutamate-induced oxytosis in HT-22 cells through GSH related as well as apoptosis-inducing factor and Bcl-2/adenovirus E1B 19 kDa-interacting protein 3-related pathways. [source] Cadmium-induced astroglial death proceeds via glutathione depletionJOURNAL OF NEUROSCIENCE RESEARCH, Issue 2 2006Joo-Young Im Abstract Cadmium is a heavy metal that accumulates in the body, and its accumulation in the brain damages both neurons and glial cells. In the current study, we explored the mechanism underlying cadmium toxicity in primary cortical astroglia cultures. Chronic treatment with 10 ,M cadmium was sufficient to cause 90% cell death in 18 hr. However, unlike that observed in neurons, cadmium-induced astroglial toxicity was not attenuated by the antioxidants trolox (100 ,M), caffeic acid (1 mM), and vitamin C (1 mM). In contrast, extracellular 100 ,M glutathione (GSH; ,-Glu-Cys-Gly) or 100 ,M cysteine almost completely blocked cadmium-induced astroglial death, whereas 300 ,M oxidized GSH (GSSG) or 300 ,M cystine, which do not have the free thiol group, were ineffective. In addition, cadmium toxicity was noticeably inhibited or enhanced when intracellular GSH was, respectively, increased by using the cell-permeable glutathione ethyl ester (GSH-EE) or depleted by using buthionine sulfoximine (BSO), an inhibitor of ,-glutamylcysteine synthetase. In agreement with these data, intracellular GSH levels were found to be depressed in cadmium-treated astrocytes. These results suggest that the toxic effect of cadmium on primary astroglial cells involves GSH depletion and, furthermore, that GSH administration can potentially be used to counteract cadmium-induced astroglial cell death therapeutically. © 2005 Wiley-Liss, Inc. [source] ,-glutamylcysteine ethyl ester-induced up-regulation of glutathione protects neurons against A,(1,42)-mediated oxidative stress and neurotoxicity: Implications for Alzheimer's diseaseJOURNAL OF NEUROSCIENCE RESEARCH, Issue 5 2005Debra Boyd-Kimball Abstract Glutathione (GSH) is an important endogenous antioxidant found in millimolar concentrations in the brain. GSH levels have been shown to decrease with aging. Alzheimer's disease (AD) is a neurodegenerative disorder associated with aging and oxidative stress. A,(1,42) has been shown to induce oxidative stress and has been proposed to play a central role in the oxidative damage detected in AD brain. It has been shown that administration of ,-glutamylcysteine ethyl ester (GCEE) increases cellular levels of GSH, circumventing the regulation of GSH biosynthesis by providing the limiting substrate. In this study, we evaluated the protective role of up-regulation of GSH by GCEE against the oxidative and neurotoxic effects of A,(1,42) in primary neuronal culture. Addition of GCEE to neurons led to an elevated mean cellular GSH level compared with untreated control. Inhibition of ,-glutamylcysteine synthetase by buthionine sulfoximine (BSO) led to a 98% decrease in total cellular GSH compared with control, which was returned to control levels by addition of GCEE. Taken together, these results suggest that GCEE up-regulates cellular GSH levels which, in turn, protects neurons against protein oxidation, loss of mitochondrial function, and DNA fragmentation induced by A,(1,42). These results are consistent with the notion that up-regulation of GSH by GCEE may play a viable protective role in the oxidative and neurotoxicity induced by A,(1,42) in AD brain. © 2005 Wiley-Liss, Inc. [source] Enhanced glutathione production by using low-pH stress coupled with cysteine addition in the treatment of high cell density culture of Candida utilisLETTERS IN APPLIED MICROBIOLOGY, Issue 5 2008G. Liang Abstract Aims:, To investigate the effects of pH stress coupled with cysteine addition on glutathione (GSH) production in the treatment of high cell density culture of Candida utilis. Methods and Results:, We have previously observed that most Candida utilis cells remained viable after being subjected to pH at 1·2 for 3 h and that some intracellular GSH leaked into the medium. A cysteine addition strategy was applied in fed-batch production of GSH. A single cysteine addition resulted in higher GSH yield than two separate additions without pH stress. An increase in intracellular GSH content triggered inhibition of ,-glutamylcysteine synthetase (,-GCS). A strategy that combines cysteine addition with low-pH stress was developed to relieve the inhibition of ,-GCS. Conclusion:, Without pH stress, single shot and double shot cysteine addition yielded a total GSH of 1423 and 1325 mg l,1. In comparison, a low-pH stress counterpart resulted in a total GSH of 1542 and 1730 mg l,1, respectively. With low-pH stress, we observed GSH secretion into the medium at 673 and 558 mg l,1 and an increase in the ,-GCS activity by 1·2- and 1·5-fold, respectively. The specific GSH production yield increased from 1·76% to 1·91% (w/w) for single shot, and 1·64% to 2·14% for double shots. Significance and Impact of the Study:, Low-pH shift was applied to alleviate the feedback inhibition of intracellular GSH on ,-GCS activity by secreting GSH into the medium. This strategy is coupled with cysteine addition to enhance GSH production in Candida utilis. [source] Importance of glutathione in the nodulation process of peanut (Arachis hypogaea)PHYSIOLOGIA PLANTARUM, Issue 2 2008Eliana Bianucci GSH appears to be essential for proper development of the root nodules during the symbiotic association of legume,rhizobia in which the entry of rhizobia involves the formation of infection threads. In the particular case of peanut,rhizobia symbiosis, the entry of rhizobia occurs by the mechanism of infection called ,crack entry', i.e. entry at the point of emergence of lateral roots. We have previously shown the role of GSH content of Bradyrhizobium sp. SEMIA 6144 during the symbiotic association with peanut using a GSH-deficient mutant obtained by disruption of the gshA gene, encoding ,-glutamylcysteine synthetase (,-GCS), which was able to induce nodules in peanut roots without alterations in the symbiotic phenotype. To investigate the role of the peanut GSH content in the symbiosis, the compound l -buthionine-sulfoximine (BSO), a specific inhibitor of ,-GCS in plants, was used. There were no differences in the plant growth and the typical anatomic structure of the peanut roots when the plants grew in the Fahraeus medium either in presence or in absence of 0.1 mM BSO. However, the GSH content was reduced by 51% after treatment with BSO. The BSO-treated plants inoculated with wild-type or mutant strains of Bradyrhizobium sp. showed a significant reduction in the number and dry weight of nodules, suggesting that GSH content could play an important role in the nodulation process of root peanut with Bradyrhizobium sp. [source] Evaluation of Transgenic Poplars Over-Expressing Enzymes of Glutathione Synthesis for Phytoremediation of CadmiumPLANT BIOLOGY, Issue 6 2002A. Koprivova Abstract: Recently, phytoremediation of soils polluted with heavy metals has received a lot of attention. Since glutathione (GSH) and its derivatives (e.g., phytochelatins) play a major role in plant defence against environmental pollutants, we tested the effects of over-expression of bacterial genes for GSH synthesis in poplar on cadmium accumulation. A pilot experiment with CdCl2 in hydroponics revealed that poplars over-expressing ,-glutamylcysteine synthetase (,-ECS) accumulated significantly more Cd in root tissue than wild type or glutathione synthetase over-expressing poplars. To test the partitioning of Cd in different organs, poplar lines over-expressing ,-ECS in the cytosol and in chloroplasts were treated with 0.2 mM CdCl2 in hydroponics. Significant amounts of Cd were translocated to leaves, but significant differences in Cd accumulation were not observed between transgenic and wild type plants. To evaluate these lines for large-scale phytoremediation of cadmium, plants were treated with 2 mM Cd in soil. Over a four-week period, the poplar plants were able to accumulate up to 5.3 mg Cd. Most remarkably, in young leaves of both transgenic lines, Cd was accumulated to concentrations 2.5 - 3 times higher than in the wild type. The increased allocation of cadmium to the young leaves represents a potentional advantage for the phytoremediation process using the same plants over several vegetation periods. The use of transgenic poplar lines with enhanced glutathione production capacity seems to be of particular advantage in highly polluted soils. [source] Role of selenium in regulation of spermatogenesis: Involvement of activator protein 1BIOFACTORS, Issue 3 2005Sonia Shalini Abstract Selenium (Se) is involved in the process of male reproduction. Several studies have been carried out to find the mechanism of Se action through identified selenoproteins. Especially selenoenzyme phospholipid glutathione peroxidase (PHGPx, GPx-4) plays a pivotal role in regulating spermatogenesis. However, the action of selenium is best known as an antioxidant which acts through various selenoproteins viz. glutathione peroxidase, thioredoxin reductase and selenoprotein P. Oxidative stress is currently being considered a leading cause of male infertility. Presently, the involvement of redox active transcription factor, AP1 (Activator protein1) in testicular function was studied. AP1 is redox sensitive and also controls cell proliferation. The effects of Se might be mediated through it. Different Se status - deficient, adequate and excess Se - were generated in male Balb/c mice by feeding yeast based selenium deficient diet and deficient diet supplemented with Se as sodium selenite (0.2 and 1 ppm Se), respectively, for a period of 4 and 8 weeks. Se status was checked by measuring the Se levels and glutathione peroxidase (GSH-Px) activity in testis and liver. The reproductive potential of mice was affected at these changed Se levels. Changes in the activity of superoxide dismutase (SOD), levels of reduced glutathione (GSH) and oxidized glutathione (GSSG) were observed indicating increased oxidative stress at both the levels. Further, changes in the mRNA expression of GSH-Px, ,-glutamylcysteine synthetase ,GCS) and Mn superoxide dismutase (MnSOD) were observed. Decrease in cjun and cfos mRNA levels were observed at both the Se status (deficient and excess) which might be responsible for decreased germ cell number, differentiation and reduced fertility observed at the altered Se levels. [source] Crystallization and preliminary crystallographic analysis of bifunctional ,-glutamylcysteine synthetase,glutatione synthetase from Streptococcus agalactiaeACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2009Yasunori Nakashima ,-Glutamylcysteine synthetase,glutathione synthetase (,GCS-GS) is a bifunctional enzyme that catalyzes two consecutive steps of ATP-dependent peptide formation in glutathione biosynthesis. Streptococcus agalactiae,GCS-GS is a target for the development of potential therapeutic agents. ,GCS-GS was crystallized using the sitting-drop vapour-diffusion method. The crystals grew to dimensions of 0.3 × 0.2 × 0.2,mm under reducing conditions with 5,mM TCEP. X-ray data were collected to 2.8,Å resolution from a tetragonal crystal that belonged to space group I41. [source] Augmented biosynthesis of cadmium sulfide nanoparticles by genetically engineered Escherichia coliBIOTECHNOLOGY PROGRESS, Issue 5 2009Yen-Lin Chen Abstract Microorganisms can complex and sequester heavy metals, rendering them promising living factories for nanoparticle production. Glutathione (GSH) is pivotal in cadmium sulfide (CdS) nanoparticle formation in yeasts and its synthesis necessitates two enzymes: ,-glutamylcysteine synthetase (,-GCS) and glutathione synthetase (GS). Hereby, we constructed two recombinant E. coli ABLE C strains to over-express either ,-GCS or GS and found that ,-GCS over-expression resulted in inclusion body formation and impaired cell physiology, whereas GS over-expression yielded abundant soluble proteins and barely impeded cell growth. Upon exposure of the recombinant cells to cadmium chloride and sodium sulfide, GS over-expression augmented GSH synthesis and ameliorated CdS nanoparticles formation. The resultant CdS nanoparticles resembled those from the wild-type cells in size (2,5 nm) and wurtzite structures, yet differed in dispersibility and elemental composition. The maximum particle yield attained in the recombinant E. coli was ,2.5 times that attained in the wild-type cells and considerably exceeded that achieved in yeasts. These data implicated the potential of genetic engineering approach to enhancing CdS nanoparticle biosynthesis in bacteria. Additionally, E. coli -based biosynthesis offers a more energy-efficient and eco-friendly method as opposed to chemical processes requiring high temperature and toxic solvents. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] | ||||||||||||||||||||||