Glutathione degradation is a key determinant of glutathione homeostasis.
ABSTRACT: Glutathione (GSH) has several important functions in eukaryotic cells, and its intracellular concentration is tightly controlled. Combining mathematical models and (35)S labeling, we analyzed Saccharomyces cerevisiae sulfur metabolism. This led us to the observation that GSH recycling is markedly faster than previously estimated. We set up additional in vivo assays and concluded that under standard conditions, GSH half-life is around 90 min. Sulfur starvation and growth with GSH as the sole sulfur source strongly increase GSH degradation, whereas cadmium (Cd(2+)) treatment inhibits GSH degradation. Whatever the condition tested, GSH is degraded by the cytosolic Dug complex (composed of the three subunits Dug1, Dug2, and Dug3) but not by the ?-glutamyl-transpeptidase, raising the question of the role of this enzyme. In vivo, both DUG2/3 mRNA levels and Dug activity are quickly induced by sulfur deprivation in a Met4-dependent manner. This suggests that Dug activity is mainly regulated at the transcriptional level. Finally, analysis of dug2? and dug3? mutant cells shows that GSH degradation activity strongly impacts on GSH intracellular concentration and that GSH intracellular concentration does not affect GSH synthesis rate. Altogether, our data led us to reconsider important aspects of GSH metabolism, challenging notions on GSH synthesis and GSH degradation that were considered as established.
Project description:Candida albicans lacks the ability to survive within its mammalian host in the absence of endogenous glutathione biosynthesis. To examine the ability of this yeast to utilize exogenous glutathione, we exploited the organic sulfur auxotrophy of C. albicans met15? strains. We observed that glutathione is utilized efficiently by the alternative pathway of glutathione degradation (DUG pathway). The major oligopeptide transporters OPT1-OPT5 of C. albicans that were most similar to the known yeast glutathione transporters were not found to contribute to glutathione transport to any significant extent. A genomic library approach to identify the glutathione transporter of C. albicans yielded OPT7 as the primary glutathione transporter. Biochemical studies on OPT7 using radiolabeled GSH uptake revealed a K(m) of 205 ?m, indicating that it was a high affinity glutathione transporter. OPT7 is unusual in several aspects. It is the most remote member to known yeast glutathione transporters, lacks the two highly conserved cysteines in the family that are known to be crucial in trafficking, and also has the ability to take up tripeptides. The transporter was regulated by sulfur sources in the medium. OPT7 orthologues were prevalent among many pathogenic yeasts and fungi and formed a distinct cluster quite remote from the Saccharomyces cerevisiae HGT1 glutathione transporter cluster. In vivo experiments using a systemic model of candidiasis failed to detect expression of OPT7 in vivo, and strains disrupted either in the degradation (dug3?) or transport (opt7?) of glutathione failed to show a defect in virulence.
Project description:We analyze the transcriptome of Ogataea parapolymorpha wild-type and met4 mutant under sulfur limited condition to investigate the function of Met4 protein in this yeast in the absence of any sulfur source. By sequencing the mRNAs of the wild-type and mutant grown in sulfur-limited B minimal medium with or without cysteine as a sole sulfur source, we find the role of OpMet4p as a master regulator for cell homeostasis under sulfur limitation. Overall design: Examination of transcriptome in Ogataea parapolymorpha wild-type and met4 mutant in the absence or presence of sulfur source
Project description:Glutathione (GSH) is a major antioxidant in cells, and plays vital roles in the cellular defense against oxidants and in the regulation of redox signals. In a previous report, we demonstrated that stem cell function is critically affected by heterogeneity and dynamic changes in cellular GSH concentration. Here, we present a detailed protocol for the monitoring of GSH concentration in living stem cells using FreSHtracer, a real-time GSH probe. We describe the steps involved in monitoring GSH concentration in single living stem cells using confocal microscopy and flow cytometry. These methods are simple, rapid, and quantitative, and able to demonstrate intracellular GSH concentration changes in real time. We also describe the application of FreSHtracer to the sorting of stem cells according to their GSH content using flow cytometry. Typically, microscopic or flow cytometric analyses of FreSHtracer and MitoFreSHtracer signals in living stem cells take ~2~3 h, and the fractionation of stem cells into subpopulations on the basis of cellular GSH levels takes 3~4.5 h. This method could be applied to almost every kind of mammalian cell with minor modifications to the protocol described here.
Project description:Glutathione (GSH) was discovered in yeast cells in 1888. Studies of GSH in mammalian cells before the 1980s focused exclusively on its function for the detoxication of xenobiotics or for drug metabolism in the liver, in which GSH is present at its highest concentration in the body. Increasing evidence has demonstrated other important roles of GSH in the brain, not only for the detoxication of xenobiotics but also for antioxidant defense and the regulation of intracellular redox homeostasis. GSH also regulates cell signaling, protein function, gene expression, and cell differentiation/proliferation in the brain. Clinically, inborn errors in GSH-related enzymes are very rare, but disorders of GSH metabolism are common in major neurodegenerative diseases showing GSH depletion and increased levels of oxidative stress in the brain. GSH depletion would precipitate oxidative damage in the brain, leading to neurodegenerative diseases. This review focuses on the significance of GSH function, the synthesis of GSH and its metabolism, and clinical disorders of GSH metabolism. A potential approach to increase brain GSH levels against neurodegeneration is also discussed.
Project description:Cell adaptation to the environment often involves induction of complex gene expression programs under the control of specific transcriptional activators. For instance, in response to cadmium, budding yeast induces transcription of the sulfur amino acid biosynthetic genes through the basic-leucine zipper activator Met4, and also launches a program of substitution of abundant glycolytic enzymes by isozymes with a lower content in sulfur. We demonstrate here that transcriptional induction of PDC6, which encodes a pyruvate decarboxylase isoform with low sulfur content, is directly controlled by Met4 and its DNA-binding cofactors the basic-helix-loop-helix protein Cbf1 and the two homologous zinc finger proteins Met31 and Met32. Study of Cbf1 and Met31/32 association with PDC6 allowed us to find a new mechanism of recruitment of Met4, which allows PDC6 being differentially regulated compared to sulfur amino acid biosynthetic genes. Our findings provide a new example of mechanism allowing transcriptional plasticity within a regulatory network thanks to a definite toolbox comprising a unique master activator and several dedicated DNA-binding cofactors. We also show evidence suggesting that integration of PDC6 to the Met4 regulon may have occurred recently in the evolution of the Saccharomyces cerevisiae lineage.
Project description:The recently identified, fungi-specific alternative pathway of glutathione degradation requires the participation of three genes, DUG1, DUG2, and DUG3. Dug1p has earlier been shown to function as a Cys-Gly-specific dipeptidase. In the present study, we describe the characterization of Dug2p and Dug3p. Dug3p has a functional glutamine amidotransferase (GATase) II domain that is catalytically important for glutathione degradation as demonstrated through mutational analysis. Dug2p, which has an N-terminal WD40 and a C-terminal M20A peptidase domain, has no peptidase activity. The previously demonstrated Dug2p-Dug3p interaction was found to be mediated through the WD40 domain of Dug2p. Dug2p was also shown to be able to homodimerize, and this was mediated by its M20A peptidase domain. In vitro reconstitution assays revealed that Dug2p and Dug3p were required together for the cleavage of glutathione into glutamate and Cys-Gly. Purification through gel filtration chromatography confirmed the formation of a Dug2p-Dug3p complex. The functional complex had a molecular weight that corresponded to (Dug2p-Dug3p)(2) in addition to higher molecular weight oligomers and displayed Michaelis-Menten kinetics. (Dug2p-Dug3p)(2) had a K(m) for glutathione of 1.2 mm, suggesting a novel GATase enzyme that acted on glutathione. Dug1p activity in glutathione degradation was found to be restricted to its Cys-Gly peptidase activity, which functioned downstream of the (Dug2p-Dug3p)(2) GATase. The DUG2 and DUG3 genes, but not DUG1, were derepressed by sulfur limitation. Based on these studies and the functioning of GATases, a mechanism is proposed for the functioning of the Dug proteins in the degradation of glutathione.
Project description:Both prokaryotic and eukaryotic organisms possess mechanisms for the detoxification of heavy metals, and these mechanisms are found among distantly related species. We investigated the role of intracellular glutathione (GSH), which, in a large number of taxa, plays a role in protection against the toxicity of common heavy metals. Anaerobically grown Lactococcus lactis containing an inducible GSH synthesis pathway was used as a model organism. Its physiological condition allowed study of putative GSH-dependent uranyl detoxification mechanisms without interference from additional reactive oxygen species. By microcalorimetric measurements of metabolic heat during cultivation, it was shown that intracellular GSH attenuates the toxicity of uranium at a concentration in the range of 10 to 150 ?M. In this concentration range, no effect was observed with copper, which was used as a reference for redox metal toxicity. At higher copper concentrations, GSH aggravated metal toxicity. Isothermal titration calorimetry revealed the endothermic binding of U(VI) to the carboxyl group(s) of GSH rather than to the reducing thiol group involved in copper interactions. The data indicate that the primary detoxifying mechanism is the intracellular sequestration of carboxyl-coordinated U(VI) into an insoluble complex with GSH. The opposite effects on uranyl and on copper toxicity can be related to the difference in coordination chemistry of the respective metal-GSH complexes, which cause distinct growth phase-specific effects on enzyme-metal interactions.Understanding microbial metal resistance is of particular importance for bioremediation, where microorganisms are employed for the removal of heavy metals from the environment. This strategy is increasingly being considered for uranium. However, little is known about the molecular mechanisms of uranyl detoxification. Existing studies of different taxa show little systematics but hint at a role of glutathione (GSH). Previous work could not unequivocally demonstrate a GSH function in decreasing the presumed uranyl-induced oxidative stress, nor could a redox-independent detoxifying action of GSH be identified. Combining metabolic calorimetry with cell number-based assays and genetics analysis enables a novel and general approach to quantify toxicity and relate it to molecular mechanisms. The results show that GSH-expressing microorganisms appear advantageous for uranyl bioremediation.
Project description:Transcription activation of sulfur metabolism in yeast is dependent on two DNA binding factors, the centromere binding factor 1 (Cbf1) and Met4. While the role of Met4 was clearly established by showing that it acts as a transcription activator, the precise function in transcription of the multi-functional factor Cbf1 remains more elusive. We report here the identification of a new transcription factor Met28 which participates in the regulation of sulfur metabolism. Cloning and sequencing of MET28 revealed that it encodes a new member of the basic leucine zipper DNA binding factor family. We also demonstrate that Met28 possesses no intrinsic transcription activation capabilities. Studies of the DNA binding characteristics of Met28 led us to identify in gel mobility assays a heteromeric complex containing Cbf1, Met4 and Met28. We further demonstrated that the presence of Cbf1 and Met4 stimulates the binding of Met28 to DNA. 'Two-hybrid' studies allowed us to carry out preliminary investigations on the binary protein-protein interactions involved in the formation of the Cbf1-Met4-Met28 complex. Our results give evidence that the leucine zippers of Met4 and Met28, along with the basic helix-loop-helix domain of Cbf1, provide the protein surfaces mediating these interactions. All these results suggest that the multi-functional factor Cbf1 functions in transcription activation by tethering specific activating factors to the DNA.
Project description:Redox-responsive polyplexes represent a promising class of non-viral gene delivery vectors. The reducible disulfide bonds in the polyplexes undergo intracellular reduction owing to the presence of high concentrations of reduced glutathione (GSH). Available evidence suggests improved transfection activity of redox-sensitive polyplexes upon artificial modulation of intracellular GSH. This study investigates the effect of innate differences in GSH concentration in a panel of human pancreatic cancer cell lines on activity of reducible polyplexes of the four major classes of nucleic acid therapeutics: plasmid DNA (pDNA), messenger RNA (mRNA), antisense oligodeoxynucleotides (AON) and siRNA. In general, reducible polyplexes of linear poly(amido amines) (PAA) show improved activity compared to non-reducible polyplexes of PAA. Results demonstrate that increased GSH levels are associated with improved transfection of mRNA polyplexes but no clear trend is observed for pDNA, AON and siRNA polyplexes.
Project description:<h4>Background</h4>In vitro and rodent studies have shown that arsenic (As) exposure can deplete glutathione (GSH) and induce oxidative stress. GSH is the primary intracellular antioxidant; it donates an electron to reactive oxygen species, thus producing glutathione disulfide (GSSG). Cysteine (Cys) and cystine (CySS) are the predominant thiol/disulfide redox couple found in human plasma. Arsenic, GSH, and Cys are linked in several ways: a) GSH is synthesized via the transsulfuration pathway, and Cys is the rate-limiting substrate; b) intermediates of the methionine cycle regulate both the transsulfuration pathway and As methylation; c) GSH serves as the electron donor for reduction of arsenate to arsenite; and d) As has a high affinity for sulfhydryl groups and therefore binds to GSH and Cys.<h4>Objectives</h4>We tested the hypothesis that As exposure is associated with decreases in GSH and Cys and increases in GSSG and CySS (i.e., a more oxidized environment).<h4>Methods</h4>For this cross-sectional study, the Folate and Oxidative Stress Study, we recruited a total of 378 participants from each of five water As concentration categories: < 10 (n = 76), 10-100 (n = 104), 101-200 (n = 86), 201-300 (n = 67), and > 300 µg/L (n = 45). Concentrations of GSH, GSSG, Cys, and CySS were measured using HPLC.<h4>Results</h4>An interquartile range (IQR) increase in water As was negatively associated with blood GSH (mean change, -25.4 µmol/L; 95% CI: -45.3, -5.31) and plasma CySS (mean change, -3.00 µmol/L; 95% CI: -4.61, -1.40). We observed similar associations with urine and blood As. There were no significant associations between As exposure and blood GSSG or plasma Cys.<h4>Conclusions</h4>The observed associations are consistent with the hypothesis that As may influence concentrations of GSH and other nonprotein sulfhydryls through binding and irreversible loss in bile and/or possibly in urine.