Expression of human glutathione S-transferases in Saccharomyces cerevisiae confers resistance to the anticancer drugs adriamycin and chlorambucil.
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ABSTRACT:
SUBMITTER: Black SM
PROVIDER: S-EPMC1131433 | biostudies-other | 1990 Jun
REPOSITORIES: biostudies-other
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Project description: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.
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Project description:Sodium dodecyl sulfate is a detergent that disrupts cell membranes, activates cell wall integrity signaling and restricts cell growth in Saccharomyces cerevisiae. However, the underlying mechanism of how sodium dodecyl sulfate inhibits cell growth is not fully understood. Previously, we have shown that deletion of the MCK1 gene leads to sensitivity to sodium dodecyl sulfate; thus, we implemented a suppressor gene screening revealing that the overexpression of TAT2 tryptophan permease rescues cell growth in sodium dodecyl sulfate-treated Δmck1 cells. Therefore, we questioned the involvement of tryptophan in the response to sodium dodecyl sulfate treatment. In this work, we show that trp1-1 cells have a disadvantage in the response to sodium dodecyl sulfate compared to auxotrophy for adenine, histidine, leucine or uracil when cells are grown on rich media. While also critical in the response to tea tree oil, TRP1 does not avert growth inhibition due to other cell wall/membrane perturbations that activate cell wall integrity signaling such as Calcofluor White, Congo Red or heat stress. This implicates a distinction from the cell wall integrity pathway and suggests specificity to membrane stress as opposed to cell wall stress. We discovered that tyrosine biosynthesis is also essential upon sodium dodecyl sulfate perturbation whereas phenylalanine biosynthesis appears dispensable. Finally, we observe enhanced tryptophan import within minutes upon exposure to sodium dodecyl sulfate indicating that these cells are not starved for tryptophan. In summary, we conclude that internal concentration of tryptophan and tyrosine makes cells more resistant to detergent such as sodium dodecyl sulfate.
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Project description:Glutathione (GSH) production during wine fermentation is a desirable trait as it can limit must and wine oxidation and protect various aromatic compounds. UMCC 2581 is a Saccharomyces cerevisiae wine strain with enhanced GSH content at the end of wine fermentation. This strain was previously derived by selection for molybdate resistance following a sexual cycle of UMCC 855 using an evolution-based strategy. In this study, we examined genetic and gene expression changes associated with the derivation of UMCC 2581. For genetic analysis we sporulated the diploid UMCC 855 parental strain and found four phenotype classes of segregants related to molybdate resistance, demonstrating the presence of segregating variation from the parental strain. Using bulk segregant analysis we mapped molybdate traits to two loci. By sequencing both the parental and evolved strain genomes we identified candidate mutations within the two regions as well as an extra copy of chromosome 1 in UMCC 2581. Combining the mapped loci with gene expression profiles of the evolved and parental strains we identified a number of candidate genes with genetic and/or gene expression changes that could underlie molybdate resistance and increased GSH levels. Our results provide insight into the genetic basis of GSH production relevant to winemaking and highlight the value of enhancing wine strains using existing variation present in wine strains.
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Project description:Glutathione S-transferases P1 (GSTP1) is a phase II detoxifying enzyme and increased expression of GSTP1 has been linked with acquired resistance to anti-cancer drugs. However, most anticancer drugs are not good substrates for GSTP1, suggesting that the contribution of GSTP1 to drug resistances might not be dependent on its capacity to detoxify chemicals or drugs. In the current study, we found a novel mechanism by which GSTP1 protects human breast cancer cells from adriamycin (ADR)-induced cell death and contributes to the drug resistance. GSTP1 protein level is very low in human breast cancer cell line MCF-7 but is high in ADR-resistant MCF-7/ADR cells. Under ADR treatment, MCF-7/ADR cells showed a higher autophagy level than MCF-7 cells. Overexpression of GSTP1 in MCF-7 cells by using the DNA transfection vector enhanced autophagy and down-regulation of GSTP1 through RNA interference in MCF-7/ADR cells decreased autophagy. When autophagy was prevented, GSTP1-induced ADR resistance reduced. We found that GSTP1 enhanced autophagy level in MCF-7 cells through interacting with p110α subunit of phosphatidylinositol-3-kinase (PI3K) and then inhibited PI3K/protein kinase B (AKT)/mechanistic target of rapamycin (mTOR) activity. Proline123, leucine160, and glutamine163, which located in C terminal of GSTP1, are essential for GSTP1 to interact with p110α, and the following autophagy and drug resistance regulation. Taken together, our findings demonstrate that high level of GSTP1 maintains resistance of breast cancer cells to ADR through promoting autophagy. These new molecular insights provide an important contribution to our better understanding the effect of GSTP1 on the resistance of tumors to chemotherapy.
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Project description:Glutathione transferases (GSTs) are ubiquitous enzymes that catalyze the conjugation of glutathione to various molecules. Among the 42 GSTs identified in Drosophila melanogaster, Delta and Epsilon are the largest classes, with 25 members. The Delta and Epsilon classes are involved in different functions, such as insecticide resistance and ecdysone biosynthesis. The insect GST number variability is due mainly to these classes. Thus, they are generally considered supports during the evolution for the adaptability of the insect species. To explore the link between Delta and Epsilon GST and their evolution, we analyzed the sequences using bioinformatic tools. Subgroups appear within the Delta and Epsilon GSTs with different levels of diversification. The diversification also appears in the sequences showing differences in the active site. Additionally, amino acids essential for structural stability or dimerization appear conserved in all GSTs. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed that the transcripts corresponding to these two classes are heterogeneously expressed within D. melanogaster. Some GSTs, such as GSTD1, are highly expressed in all tissues, suggesting their general function in detoxification. Conversely, some others, such as GSTD11 or GSTE4, are specifically expressed at a high level specifically in antennae, suggesting a potential role in olfaction.
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Project description:The 55 Arabidopsis glutathione transferases (GSTs) are, with one microsomal exception, a monophyletic group of soluble enzymes that can be divided into phi, tau, theta, zeta, lambda, dehydroascorbate reductase (DHAR) and TCHQD classes. The populous phi and tau classes are often highly stress inducible and regularly crop up in proteomic and transcriptomic studies. Despite much study on their xenobiotic-detoxifying activities their natural roles are unclear, although roles in defence-related secondary metabolism are likely. The smaller DHAR and lambda classes are likely glutathione-dependent reductases, the zeta class functions in tyrosine catabolism and the theta class has a putative role in detoxifying oxidised lipids. This review describes the evidence for the functional roles of GSTs and the potential for these enzymes to perform diverse functions that in many cases are not "glutathione transferase" activities. As well as biochemical data, expression data from proteomic and transcriptomic studies are included, along with subcellular localisation experiments and the results of functional genomic studies.
| S-EPMC3244946 | biostudies-literature
Project description:Adriamycin is a widely used drug for the treatment of various types of cancers, but its clinical application is limited because of irreversible dilated cardiomyopathy. The incidence of cardiomyopathy is a consequence of disrupted energy production, which could be related to the defects in glycogen, lipid and mucopolysaccharide metabolism. We explored the effect of Adriamycin on enzymes involved in glycolysis and apoptotic genes through molecular docking. We used Saccharomyces cerevisiae as model organism and studied the effect of Adriamycin on selected enzymes involved in glycolysis. The docking studies revealed that Adriamycin interacts with phosphofructokinase and enolase in an efficient manner. In phosphofructokinase, Adriamycin binds at the active site and with enolase the drug interacts at the cofactor-binding site (Mg2+) which might impair the activity of the enzyme. Gene expression studies revealed that Adriamycin causes the dysregulation of glycolysis through dysregulation of hexokinase, phosphoglycerate mutase, enolase and downregulation of pyruvate kinase. The drug shows a biphasic effect on the expression of genes enolase and pyruvate kinase. The impairment in glycolysis might reduce the ATP synthesis, and the cells might be deprived of energy. The condition is further worsened by elevated ROS levels triggering the cell to undergo apoptosis evidenced by downregulation of SOD and upregulation of BAX and caspase. In conclusion, our study reveals that Adriamycin impairs glycolysis and cause cell to undergo apoptosis due to oxidative stress in yeast cells.
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Project description:Saccharomyces cerevisiae yeast, when pretreated with elevated temperatures, undergo adaptive changes that promote survival after an otherwise lethal heat stress. The heat shock response, a cellular stress response variant, mediates these adaptive changes. Ethanol, a low-potency anesthetic, promotes thermotolerance possibly through heat shock response activation. Therefore, we hypothesized other anesthetic compounds, like ethanol, may invoke the heat shock response to promote thermotolerance. To test this hypothesis, we pretreated yeast with a series of non-volatile anesthetic and anesthetic-related compounds and quantified survival following lethal heat shock (52 °C for 5 min). Most compounds invoked thermoprotection and promoted survival with a potency proportional to hydrophobicity: tribromoethanol (5.6 mM, peak survival response), trichloroethanol (17.8 mM), dichloroethanol (100 mM), monochloroethanol (316 mM), trifluoroethanol (177.8 mM), ethanol (1 M), isopropanol (1 M), propofol (316 μM), and carbon tetrabromide (32 μM). Thermoprotection conferred by pretreatment with elevated temperatures was "left shifted" by anesthetic co-treatment from (in °C) 35.3 ± 0.1 to 32.2 ± 0.1 with trifluoroethanol (177.8 mM), to 31.2 ± 0.1 with trichloroethanol (17.8 mM), and to 29.1 ± 0.3 with tribromoethanol (5.6 mM). Yeast in postdiauxic shift growth phase, relative to mid-log, responded with greater heat shock survival; and media supplementation with tryptophan and leucine blocked thermoprotection, perhaps by reversing the amino acid starvation response. Our results suggest S. cerevisase may serve as a model organism for understanding anesthetic toxicity and anesthetic preconditioning, a process by which anesthetics promote tissue survival after hypoxic insult.
| S-EPMC8177089 | biostudies-literature
Project description:Glutathione transferase classical GSH conjugation activity plays a critical role in cellular detoxification against xenobiotics and noxious compounds as well as against oxidative stress. However, this feature is also exploited by cancer cells to acquire drug resistance and improve their survival. As a result, various members of the family were found overexpressed in a number of different cancers. Moreover several GST polymorphisms, ranging from null phenotypes to point mutations, were detected in members of the family and found to correlate with the onset of neuro-degenerative diseases. In the last decades, a great deal of research aimed at clarifying the role played by GSTs in drug resistance, at developing inhibitors to counteract this activity but also at exploiting GSTs for prodrugs specific activation in cancer cells. Here we summarize some of the most important achievements reached in this lively area of research.
| S-EPMC5833873 | biostudies-literature
Project description:SummaryThe soluble glutathione transferases (GSTs, EC 2.5.1.18) are encoded by a large and diverse gene family in plants, which can be divided on the basis of sequence identity into the phi, tau, theta, zeta and lambda classes. The theta and zeta GSTs have counterparts in animals but the other classes are plant-specific and form the focus of this article. The genome of Arabidopsis thaliana contains 48 GST genes, with the tau and phi classes being the most numerous. The GST proteins have evolved by gene duplication to perform a range of functional roles using the tripeptide glutathione (GSH) as a cosubstrate or coenzyme. GSTs are predominantly expressed in the cytosol, where their GSH-dependent catalytic functions include the conjugation and resulting detoxification of herbicides, the reduction of organic hydroperoxides formed during oxidative stress and the isomerization of maleylacetoacetate to fumarylacetoacetate, a key step in the catabolism of tyrosine. GSTs also have non-catalytic roles, binding flavonoid natural products in the cytosol prior to their deposition in the vacuole. Recent studies have also implicated GSTs as components of ultraviolet-inducible cell signaling pathways and as potential regulators of apoptosis. Although sequence diversification has produced GSTs with multiple functions, the structure of these proteins has been highly conserved. The GSTs thus represent an excellent example of how protein families can diversify to fulfill multiple functions while conserving form and structure.
| S-EPMC139027 | biostudies-literature