Menadione stress in Saccharomyces cerevisiae strains deficient in the glutathione transferases

Using S. cerevisiae as a eukaryotic cell model we have analyzed the involvement of both glutathione transferase isoforms, Gtt1 and Gtt2, in constitutive resistance and adaptive response to menadione, a quinone which can exert its toxicity as redox cycling and/or electrophiles. The detoxification properties, of these enzymes, have also been analyzed by the appearance of S-conjugates in the media. Direct exposure to menadione (20 mM/60 min) showed to be lethal for cells deficient on both Gtt1 and Gtt2 isoforms. However, after pre-treatment with a low menadione concentration, cells deficient in Gtt2 displayed reduced ability to acquire tolerance when compared with the control and the Gtt1 deficient strains. Analyzing the toxic effects of menadione we observed that the gtt2 mutant showed no reduction in lipid peroxidation levels. Moreover, measuring the levels of intracellular oxidation during menadione stress we have shown that the increase of this oxidative stress parameter was due to the capacity menadione possesses in generating reactive oxygen species (ROS) and that both GSH and Gtt2 isoform were required to enhance ROS production. Furthermore, the efflux of the menadione–GSH conjugate, which is related with detoxification of xenobiotic pathways, was not detected in the gtt2 mutant. Taken together, these results suggest that acquisition of tolerance against stress generated by menadione and the process of detoxification through S-conjugates are dependent upon Gtt2 activity. This assessment was corroborated by the increase of GTT2 expression, and not of GTT1, after menadione treatment.     

Enhanced glutathione production by evolutionary engineering of Saccharomyces cerevisiae strains

Glutathione is an important natural tripeptide mainly used because of its antioxidative properties. Commercial glutathione is microbially synthesized by yeasts and the growing demand requires the development of new production strains. An adaptive laboratory evolution strategy using acrolein as a selection agent was employed to obtain strains with an enhanced glutathione accumulation phenotype accompanied by an acrolein resistance phenotype. Two particularly interesting isolates were obtained: one with a high volumetric productivity for glutathione reaching 8.3 mg_glutathione/L h, which is twice as high as the volumetric productivity of its parental strain. This strain reached an elevated intracellular glutathione content of 3.9%. A second isolate with an even higher acrolein tolerance exhibited a lower volumetric productivity of 5.8 mg_glutathione/L h due to a growth phenotype. However, this evolved strain accumulated glutathione in 3.3‐fold higher concentration compared to its parental strain and reached a particularly high glutathione content of almost 6%. The presented results demonstrate that acrolein is a powerful selection agent to obtain high glutathione accumulation strains in an adaptive laboratory evolution experiment.     

Oxidative stress resistance of Escherichia coli strains deficient in glutathione biosynthesis

Sensitivity to various oxidants was determined for Escherichia coli strains JTG10 and 821 deficient in biosynthesis of glutathione (gsh-) and their common parental strain AB1157 (gsh+). The three strains showed identical sensitivity to H2O2. E. coli 821 was more resistant than AB1157 and JTG10 to menadione, cumene hydroperoxide, and N-ethylmaleimide. This resistance was not related to the gsh mutation because the other gsh- mutant and the parental strain showed similar sensitivity to these oxidants. The measured activities of NADPH:menadione diaphorase and glucose-6-phosphate dehydrogenase and the extracellular level of menadione suggested that the enhanced resistance of E. coli 821 to menadione might be due to decreased diaphorase activity, but not to a lowered rate of menadione uptake.     

Saccharomyces cerevisiae cells have three Omega class glutathione S-transferases acting as 1-Cys thiol transferases

The Saccharomyces cerevisiae genome encodes three proteins that display similarities with human GSTOs (Omega class glutathione S-transferases) hGSTO1-1 and hGSTO2-2. The three yeast proteins have been named Gto1, Gto2 and Gto3, and their purified recombinant forms are active as thiol transferases (glutaredoxins) against HED (beta-hydroxyethyl disulphide), as dehydroascorbate reductases and as dimethylarsinic acid reductases, while they are not active against the standard GST substrate CDNB (1-chloro-2,4-dinitrobenzene). Their glutaredoxin activity is also detectable in yeast cell extracts. The enzyme activity characteristics of the Gto proteins contrast with those of another yeast GST, Gtt1. The latter is active against CDNB and also displays glutathione peroxidase activity against organic hydroperoxides such as cumene hydroperoxide, but is not active as a thiol transferase. Analysis of point mutants derived from wild-type Gto2 indicates that, among the three cysteine residues of the molecule, only the residue at position 46 is required for the glutaredoxin activity. This indicates that the thiol transferase acts through a monothiol mechanism. Replacing the active site of the yeast monothiol glutaredoxin Grx5 with the proposed Gto2 active site containing Cys46 allows Grx5 to retain some activity against HED. Therefore the residues adjacent to the respective active cysteine residues in Gto2 and Grx5 are important determinants for the thiol transferase activity against small disulphide-containing molecules.     

Structural homology among mammalian and Saccharomyces cerevisiae isoprenyl-protein transferases

Farnesyl-protein transferase (FTase) purified from rat or bovine brain is an alpha/beta heterodimer, comprised of subunits having relative molecular masses of approximately 47 (alpha) and 45 kDa (beta). In the yeast Saccharomyces cerevisiae, two unlinked genes, RAM1/DPR1 (RAM1) and RAM2, are required for FTase activity. To explore the relationship between the mammalian and yeast enzymes, we initiated cloning and immunological analyses. cDNA clones encoding the 329-amino acid COOH-terminal domain of bovine FTase alpha-subunit were isolated. Comparison of the amino acid sequences deduced from the alpha-subunit cDNA and the RAM2 gene revealed 30% identity and 58% similarity, suggesting that the RAM2 gene product encodes a subunit for the yeast FTase analogous to the bovine FTase alpha-subunit. Antisera raised against the RAM1 gene product reacted specifically with the beta-subunit of bovine FTase, suggesting that the RAM1 gene product is analogous to the bovine FTase beta-subunit. Whereas a ram1 mutation specifically inhibits FTase, mutations in the CDC43 and BET2 genes, both of which are homologous to RAM1, specifically inhibit geranylgeranyl-protein transferase (GGTase) type I and GGTase-II, respectively. In contrast, a ram2 mutation impairs both FTase and GGTase-I, but has little effect on GGTase-II. Antisera that specifically recognized the bovine FTase alpha-subunit precipitated both bovine FTase and GGTase-I activity, but not GGTase-II activity. Together, these results indicate that for both yeast and mammalian cells, FTase, GGTase-I, and GGTase-II are comprised of different but homologous beta-subunits and that the alpha-subunits of FTase and GGTase-I share common features not shared by GGTase-II.     

Antioxidant activity of L-ascorbic acid in wild-type and superoxide dismutase deficient strains of Saccharomyces cerevisiae

Much has been published on the non-enzymatic antioxidant L-ascorbic acid (vitamin C), but even so its interaction with endogenous cellular defense systems has not yet been fully elucidated. Our study investigated the antioxidant activity of L-ascorbic acid in wild-type strain EG103 (SOD) Saccharomyces cerevisiae and isogenic mutant strains deficient in cytosolic superoxide dismutase (sod1delta), mitochondrial superoxide dismutase (sod2delta) or both (sod1delta sod2delta), metabolizing aerobically or anaerobically with and without the stressing agent paraquat. The results show that during both aerobic and anaerobic metabolism there was a significant increase in the survival of both wild-type S. cerevisiae cells and the mutant cells (sod1delta, sod2delta and sod1delta sod2delta) when pretreated with L-ascorbic acid before exposure to paraquat. Exposure to paraquat resulted in higher catalase activity but this significantly decreased when the cells were pre-treated with L-ascorbic acid. These results demonstrate that due to the damage caused by paraquat, the antioxidant protection of L-ascorbic acid seems to be mediated by catalase levels in yeast cells.     

Genetic analysis of glutathione peroxidase in oxidative stress response of Saccharomyces cerevisiae.

Three glutathione peroxidase homologs (YKL026C, YBR244W, and YIR037W/HYR1) were found in the Saccharomyces Genome Database. We named them GPX1, GPX2, and GPX3, respectively, and we investigated the function of each gene product. The gpx3Delta mutant was hypersensitive to peroxides, whereas null mutants of the GPX1 and GPX2 did not show any obvious phenotypes. Glutathione peroxidase activity decreased approximately 57 and 93% in the gpx3Delta and gpx1Delta/gpx2Delta/gpx3Delta mutants, respectively, compared with that of wild type. Expression of the GPX3 gene was not induced by any stresses tested, whereas that of the GPX1 gene was induced by glucose starvation. The GPX2 gene expression was induced by oxidative stress, which was dependent upon the Yap1p. The TSA1 (thiol-specific antioxidant) gene encodes thioredoxin peroxidase that can reduce peroxides by using thioredoxin as a reducing power. Disruption of the TSA1 gene enhanced the basal expression level of the Yap1p target genes such as GSH1, GLR1, and GPX2 and that resulted in increases of total glutathione level and activities of glutathione reductase and glutathione peroxidase. However, expression of the TSA1 gene did not increase in the gpx1Delta/gpx2Delta/gpx3Delta mutant. Therefore, de novo synthesis and recycling of glutathione were increased in the tsa1Delta mutant to maintain the catalytic cycle of glutathione peroxidase reaction efficiently as a backup system for thioredoxin peroxidase.     

Mutants of Saccharomyces cerevisiae deficient in adenine phosphoribosyltransferase

Spontaneous and ethyl methanesulfate induced mutants of Saccharomyces cerevisiae, with partial and complete deficiency of adenine phosphoribosyltransferase (APRT, EC 2.4.2.7), were isolated by selection for resistance to 8-azaadenine. Matings between totally deficient mutants and tester strain resulted in diploid heterozygotes that were sensitive to azaadenine. Upon sporulation and tetrad analysis, azaadenine resistance (and APRT deficiency) segregated as expected for a single Mendelian gene. Hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) activity in the mutants was similar to that in the wild-type cells. There was no detectable activity of adenine aminohydrolase (EC 3.5.4.2) in the wild-type or mutant cells.     

Catalase T deficient mutants of Saccharomyces cerevisiae

A method for the isolation of catalase T deficient mutants of Saccharomyces cerevisiae is described. Ten mutants lacking catalase T and belonging to 5 complementation groups were isolated. CTT1 locus was identified as the structural gene for catalase T. It is under the control of CTT2, CTT3 and CTT4 loci.     

Differential regulation of the N-methyl transferases responsible for phosphatidylcholine synthesis in Saccharomyces cerevisiae

The enzymes catalyzing the conversion of phosphatidylethanolamine to phosphatidylcholine were assayed by measuring the incorporation of label from [¹⁴C-CH₃]-S-adenosyl-methionine into the endogenous phospholipids of particulate, cell-free preparations from S. cerevisiae grown in the presence of N-methylethanolamine, N,N-dimethylethanolamine, or choline. The results indicate that each base in the growth medium results in reduced levels of all the N-methyltransferase activity involved in the formation of the phosphatidyl ester of the given base. By following the conversion of exogenous [³²P]-phosphatidyldimethylethanolamine to [³²P]-phosphatidylcholine it has been shown that the activity of the third methyl transfer is 90% lower in particles prepared from choline grown cells than in particles prepared from cells grown without choline. The results suggest that there are at least two enzymes involved in the conversion of phosphatidylethanolamine to phosphatidylcholine and that their levels can be regulated individually.Supplementing the growth medium with any of the three methylated aminoethanols results in markedly increased cellular levels of their corresponding phosphatidyl esters and decreased levels of the precursor phosphatidyl esters. The fatty acid composition of phosphatidylcholine also changes when the medium is supplemented with choline suggesting that the proportions of the molecular species of this phosphatide depends on whether synthesis is via methylation of phosphatidylethanolamino or from the supplemented aminoethanol.     

Saccharomyces cerevisiae MGS1 is essential in strains deficient in the RAD6-dependent DNA damage tolerance pathway

Saccharomyces cerevisiae Mgs1 protein, which possesses DNA-dependent ATPase and single strand DNA annealing activities, plays a role in maintaining genomic stability. We found that mgs1 is synthetic lethal with rad6 and exhibits a synergistic growth defect with rad18 and rad5, which are members of the RAD6 epistasis group important for tolerance of DNA damage during DNA replication. The mgs1 mutant is not sensitive to DNA-damaging agents, but the mgs1 rad5 double mutant has increased sensitivity to hydroxyurea and a greatly increased spontaneous mutation rate. Growth defects of mgs1 rad18 double mutants are suppressed by a mutation in SRS2, encoding a DNA helicase, or by overexpression of Rad52. More over, mgs1 mutation suppresses the temperature sensitivity of mutants in POL3, encoding DNA polymerase delta. mgs1 also suppresses the growth defect of a pol3 mutant caused by expression of Escherichia coli RuvC, a bacterial Holliday junction resolvase. These findings suggest that Mgs1 is essential for preventing genome instability caused by replication fork arrest in cells deficient in the RAD6 pathway and may modulate replication fork movement catalyzed by yeast polymerase delta.     

Oxidized glutathione fermentation using Saccharomyces cerevisiae engineered for glutathione metabolism

Glutathione is a valuable tripeptide that is widely used in the pharmaceutical, food, and cosmetic industries. Intracellular glutathione exists in two forms, reduced glutathione (GSH) and oxidized glutathione (GSSG). Most of the glutathione produced by fermentation using yeast is in the GSH form because intracellular GSH concentration is higher than GSSG concentration. However, the stability of GSSG is higher than GSH, which makes GSSG more advantageous for industrial production and storage after extraction. In this study, an oxidized glutathione fermentation method using Saccharomyces cerevisiae was developed by following three metabolic engineering steps. First, over-expression of the glutathione peroxidase 3 (GPX3) gene increased the GSSG content better than over-expression of other identified peroxidase (GPX1 or GPX2) genes. Second, the increase in GSSG brought about by GPX3 over-expression was enhanced by the over-expression of the GSH1/GSH2 genes because of an increase in the total glutathione (GSH + GSSG) content. Finally, after deleting the glutathione reductase (GLR1) gene, the resulting GPX3/GSH1/GSH2 over-expressing ΔGLR1 strain yielded 7.3-fold more GSSG compared with the parental strain without a decrease in cell growth. Furthermore, use of this strain also resulted in an enhancement of up to 1.6-fold of the total glutathione content compared with the GSH1/GSH2 over-expressing strain. These results indicate that the increase in the oxidized glutathione content helps to improve the stability and total productivity of glutathione.     

Duplication processes in Saccharomyces cerevisiae haploid strains

Duplication is thought to be one of the main processes providing a substrate on which the effects of evolution are visible. The mechanisms underlying this chromosomal rearrangement were investigated here in the yeast Saccharomyces cerevisiae. Spontaneous revertants containing a duplication event were selected and analyzed. In addition to the single gene duplication described in a previous study, we demonstrated here that direct tandem duplicated regions ranging from 5 to 90 kb in size can also occur spontaneously. To further investigate the mechanisms in the duplication events, we examined whether homologous recombination contributes to these processes. The results obtained show that the mechanisms involved in segmental duplication are RAD52-independent, contrary to those involved in single gene duplication. Moreover, this study shows that the duplication of a given gene can occur in S.cerevisiae haploid strains via at least two ways: single gene or segmental duplication.     

Subcellular distribution of glutathione and its dynamic changes under oxidative stress in the yeast Saccharomyces cerevisiae

Glutathione is an important antioxidant in most prokaryotes and eukaryotes. It detoxifies reactive oxygen species and is also involved in the modulation of gene expression, in redox signaling, and in the regulation of enzymatic activities. In this study, the subcellular distribution of glutathione was studied in Saccharomyces cerevisiae by quantitative immunoelectron microscopy. Highest glutathione contents were detected in mitochondria and subsequently in the cytosol, nuclei, cell walls, and vacuoles. The induction of oxidative stress by hydrogen peroxide (H(2) O(2) ) led to changes in glutathione-specific labeling. Three cell types were identified. Cell types I and II contained more glutathione than control cells. Cell type II differed from cell type I in showing a decrease in glutathione-specific labeling solely in mitochondria. Cell type III contained much less glutathione contents than the control and showed the strongest decrease in mitochondria, suggesting that high and stable levels of glutathione in mitochondria are important for the protection and survival of the cells during oxidative stress. Additionally, large amounts of glutathione were relocated and stored in vacuoles in cell type III, suggesting the importance of the sequestration of glutathione in vacuoles under oxidative stress.     

Involvement of glutathione transferases,Gtt1and Gtt2, with oxidative stress response generated by H2O2 during growth of Saccharomyces cerevisiae

Abstract

Both type 1 and type 2 diabetes (insulin-dependent and non-insulin dependent diabetes, respectively) are associated with increased risk for microvascular and macrovascular complications including retinopathy, neuropathy, nephropathy and atherosclerosis. Type 2 diabetes markedly increases the risk for cardiovascular morbidity and mortality, which has major public health implications. In this review, molecular mechanisms pertaining to diabetes-induced heart pathology are addressed.     

Optimization of the medium for glutathione production in Saccharomyces cerevisiae

As a powerful statistical experimental design, uniform design (UD) method has been successfully applied in various fields such as fermentation industry, pharmaceuticals, and others. In this paper, UD was applied to optimize the medium composition for glutathione production in shake-flask culture of Saccharomyces cerevisiae T65. The experiments of nine factors (glucose, yeast extract, peptone, malt extract, molasses, MgSO₄, ZnSO₄, (NH₄)₂HPO₄ and thiamine) and nine levels were carried out according to the uniform design table U₂₇(9⁹). The experimental data was analyzed to obtain the regression model and the optimal medium composition was achieved by optimization with UD 3.0 software. The optimal medium consisted of 70 g/L glucose, 3 g/L yeast extract, 5 g/L peptone, 70 g/L malt extract, 20 g/L molasses, 5.6 g/L MgSO₄, 16 mg/L ZnSO₄, 7 g/L (NH₄)₂HPO₄ and 0.2 mg/L thiamine. The GSH yield at the optimal point achieved 74.6 mg/L, which was 1.81 times higher than that of the control. The application of UD method resulted in enhancement in GSH production.     

Cadmium-induced oxidative stress in Saccharomyces cerevisiae

The present study was undertaken to determine the effect of cadmium (Cd) on the antioxidant status of the yeast Saccharomyces cerevisiae. S. cerevisiae serves as a good eukaryotic model system for the study of the molecular mechanisms of oxidative stress. We investigated the adaptative response of S. cerevisiae exposed to Cd. Yeast cells could tolerate up to 100 microM Cd and an inhibition in the growth and viability was observed. Exposure of yeast cells to Cd showed an increase in malondialdehyde and glutathione. The activities of catalase, superoxide dismutase and glutathione peroxidase were also high in Cd-exposed cells. The incorporation of Cd led to significant increase in iron, zinc and inversely the calcium, copper levels were reduced. The results suggest that antioxidants were increased and are involved in the protection against macromolecular damage during oxidative stress; presumably, these enzymes are essential for counteracting the pro-oxidant effects of Cd.     

Production of glutathione in extracellular form by Saccharomyces cerevisiae

The present research was aimed at inducing, in a post fermentative procedure (biotransformation) and by modifying cell permeability, glutathione (GSH) accumulation and subsequent release from cells of Saccharomyces cerevisiae. With the aim of limiting process costs, research considered the possibility of employing baker's yeasts (S. cerevisiae), inexpensive cells source available on the market, in comparison with a collection strain. The tested yeasts showed different sensitivity to the chemical/physical treatments performed to alter cell permeability. Modest effects were evidenced with Triton, active only on Zeus yeast samples (1.7 g GSH/l, near 60% of which in extracellular form). Lauroyl sarcosine showed an interesting action on GB Italy sample (2.8 g GSH/l, near 80% extracellular). Lyophilization evidenced good performance with Lievitalia yeast strain (2.9 g GSH/l, 90% extracellular). The possibility of obtaining GSH directly in extracellular form represents an interesting opportunity of reducing GSH production cost and furthering the range of application of this molecule.