Gabriel G. Perrone

Reactive oxygen species and yeast apoptosis

Apoptosis is associated in many cases with the generation of reactive oxygen species (ROS) in cells across a wide range of organisms including lower eukaryotes such as the yeast Saccharomyces cerevisiae. Currently there are many unresolved questions concerning the relationship between apoptosis and the generation of ROS. These include which ROS are involved in apoptosis, what mechanisms and targets are important and whether apoptosis is triggered by ROS damage or ROS are generated as a consequence or part of the cellular disruption that occurs during cell death. Here we review the nature of the ROS involved, the damage they cause to cells, summarise the responses of S. cerevisiae to ROS and discuss those aspects in which ROS affect cell integrity that may be relevant to the apoptotic process.

Regulation of Protein S-Thiolation by Glutaredoxin 5 in the Yeast Saccharomyces cerevisiae

The irreversible oxidation of cysteine residues can be prevented by protein S-thiolation, a process by which protein -SH groups form mixed disulfides with low molecular weight thiols such as glutathione. We report here that this protein modification is not a simple response to the cellular redox state, since different oxidants lead to different patterns of proteinS-thiolation. SDS-polyacrylamide gel electrophoresis shows that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is the major target for modification following treatment with hydroperoxides (hydrogen peroxide or tert-butylhydroperoxide), whereas this enzyme is unaffected following cellular exposure to the thiol oxidant diamide. Further evidence that protein S-thiolation is tightly regulated in response to oxidative stress is provided by the finding that the Tdh3 GAPDH isoenzyme, and not the Tdh2 isoenzyme, isS-thiolated following exposure to H2O2 in vivo, whereas both GAPDH isoenzymes are S-thiolated when H2O2 is added to cell-free extracts. This indicates that cellular factors are likely to be responsible for the difference in GAPDH S-thiolation observed in vivo rather than intrinsic structural differences between the GAPDH isoenzymes. To begin to search for factors that can regulate theS-thiolation process, we investigated the role of the glutaredoxin family of oxidoreductases. We provide the first evidence that protein dethiolation in vivo is regulated by a monothiol-glutaredoxin rather than the classical glutaredoxins, which contain two active site cysteine residues. In particular, glutaredoxin 5 is required for efficient dethiolation of the Tdh3 GAPDH isoenzyme.

Genetic basis of arsenite and cadmium tolerance in Saccharomyces cerevisiae.

BackgroundArsenic and cadmium are widely distributed in nature and pose serious threats to the environment and human health. Exposure to these nonessential toxic metals may result in a variety of human diseases including cancer. However, arsenic and cadmium toxicity targets and the cellular systems contributing to tolerance acquisition are not fully known.ResultsTo gain insight into metal action and cellular tolerance mechanisms, we carried out genome-wide screening of the Saccharomyces cerevisiae haploid and homozygous diploid deletion mutant collections and scored for reduced growth in the presence of arsenite or cadmium. Processes found to be required for tolerance to both metals included sulphur and glutathione biosynthesis, environmental sensing, mRNA synthesis and transcription, and vacuolar/endosomal transport and sorting. We also identified metal-specific defence processes. Arsenite-specific defence functions were related to cell cycle regulation, lipid and fatty acid metabolism, mitochondrial biogenesis, and the cytoskeleton whereas cadmium-specific defence functions were mainly related to sugar/carbohydrate metabolism, and metal-ion homeostasis and transport. Molecular evidence indicated that the cytoskeleton is targeted by arsenite and that phosphorylation of the Snf1p kinase is required for cadmium tolerance.ConclusionThis study has pin-pointed core functions that protect cells from arsenite and cadmium toxicity. It also emphasizes the existence of both common and specific defence systems. Since many of the yeast genes that confer tolerance to these agents have homologues in humans, similar biological processes may act in yeast and humans to prevent metal toxicity and carcinogenesis.

The Thioredoxin-Thioredoxin Reductase System Can Function in Vivo as an Alternative System to Reduce Oxidized Glutathione in Saccharomyces cerevisiae

Cellular mechanisms that maintain redox homeostasis are crucial, providing buffering against oxidative stress. Glutathione, the most abundant low molecular weight thiol, is considered the major cellular redox buffer in most cells. To better understand how cells maintain glutathione redox homeostasis, cells of Saccharomyces cerevisiae were treated with extracellular oxidized glutathione (GSSG), and the effect on intracellular reduced glutathione (GSH) and GSSG were monitored over time. Intriguingly cells lacking GLR1 encoding the GSSG reductase in S. cerevisiae accumulated increased levels of GSH via a mechanism independent of the GSH biosynthetic pathway. Furthermore, residual NADPH-dependent GSSG reductase activity was found in lysate derived from glr1 cell. The cytosolic thioredoxin-thioredoxin reductase system and not the glutaredoxins (Grx1p, Grx2p, Grx6p, and Grx7p) contributes to the reduction of GSSG. Overexpression of the thioredoxins TRX1 or TRX2 in glr1 cells reduced GSSG accumulation, increased GSH levels, and reduced cellular glutathione Eh′. Conversely, deletion of TRX1 or TRX2 in the glr1 strain led to increased accumulation of GSSG, reduced GSH levels, and increased cellular Eh′. Furthermore, it was found that purified thioredoxins can reduce GSSG to GSH in the presence of thioredoxin reductase and NADPH in a reconstituted in vitro system. Collectively, these data indicate that the thioredoxin-thioredoxin reductase system can function as an alternative system to reduce GSSG in S. cerevisiae in vivo.

The critical role of glutathione in maintenance of the mitochondrial genome

Glutathione (GSH) is a key redox buffer and protectant. Growth (approx. one or two divisions) of cells lacking γ-glutamylcysteine synthetase (gsh1) in the absence of GSH led to irreversible respiratory incompetency in all cells, and after five divisions 75% of cells completely lacked mitochondrial DNA (mtDNA). The level of GSH required to allow continuous growth was distinct from that required to prevent loss of mtDNA. GSH limitation led to a change in the transcript levels of 190 genes, including 30 genes regulated by the Aft1p and/or Aft2p transcription factors, which regulate the cellular response to changes in iron availability. Disruption of AFT1 but not AFT2 in gsh1 cells afforded a protective effect on maintenance of respiratory competency, as did overexpression of GRX3 or GRX4 (encoding monothiol glutaredoxins that act as negative regulators of Aft1p). Importantly, an iron-independent mechanism (~30%) was also observed to mediate GSH-dependent mtDNA loss. Analysis of the redox environment in the cytosol, mitochondrial matrix, and intermembrane space (IMS) found that the cytosol was most severely and rapidly affected by GSH depletion. GSH may also modulate the redox environment of the IMS. The implications of altered GSH homeostasis for maintenance of mtDNA, compartmental redox, and the pathophysiology of certain diseases are discussed.

Thioredoxins function as deglutathionylase enzymes in the yeast Saccharomyces cerevisiae

BackgroundProtein-SH groups are amongst the most easily oxidized residues in proteins, but irreversible oxidation can be prevented by protein glutathionylation, in which protein-SH groups form mixed disulphides with glutathione. Glutaredoxins and thioredoxins are key oxidoreductases which have been implicated in regulating glutathionylation/deglutathionylation in diverse organisms. Glutaredoxins have been proposed to be the predominant deglutathionylase enzymes in many plant and mammalian species, whereas, thioredoxins have generally been thought to be relatively inefficient in deglutathionylation.ResultsWe show here that the levels of glutathionylated proteins in yeast are regulated in parallel with the growth cycle, and are maximal during stationary phase growth. This increase in glutathionylation is not a response to increased reactive oxygen species generated from the shift to respiratory metabolism, but appears to be a general response to starvation conditions. Our data indicate that glutathionylation levels are constitutively high in all growth phases in thioredoxin mutants and are unaffected in glutaredoxin mutants. We have confirmed that thioredoxins, but not glutaredoxins, catalyse deglutathionylation of model glutathionylated substrates using purified thioredoxin and glutaredoxin proteins. Furthermore, we show that the deglutathionylase activity of thioredoxins is required to reduce the high levels of glutathionylation in stationary phase cells, which occurs as cells exit stationary phase and resume vegetative growth.ConclusionsThere is increasing evidence that the thioredoxin and glutathione redox systems have overlapping functions and these present data indicate that the thioredoxin system plays a key role in regulating the modification of proteins by the glutathione system.

Cu, Zn superoxide dismutase and NADP(H) homeostasis are required for tolerance of endoplasmic reticulum stress in Saccharomyces cerevisiae.

Genome-wide screening for sensitivity to chronic endoplasmic reticulum (ER) stress induced by dithiothreitol and tunicamycin (TM) identified mutants deleted for Cu, Zn superoxide dismutase (SOD) function (SOD1, CCS1) or affected in NADPH generation via the pentose phosphate pathway (TKL1, RPE1). TM-induced ER stress led to an increase in cellular superoxide accumulation and an increase in SOD1 expression and Sod1p activity. Prior adaptation of the hac1 mutant deficient in the unfolded protein response (UPR) to the superoxide-generating agent paraquat reduced cell death under ER stress. Overexpression of the ER oxidoreductase Ero1p known to generate hydrogen peroxide in vitro, did not lead to increased superoxide levels in cells subjected to ER stress. The mutants lacking SOD1, TKL1, or RPE1 exhibited decreased UPR induction under ER stress. Sensitivity of the sod1 mutant to ER stress and decreased UPR induction was partially rescued by overexpression of TKL1 encoding transketolase. These data indicate an important role for SOD and cellular NADP(H) in cell survival during ER stress, and it is proposed that accumulation of superoxide affects NADP(H) homeostasis, leading to reduced UPR induction during ER stress.

Adaptation to hydrogen peroxide in Saccharomyces cerevisiae: The role of NADPH-generating systems and the SKN7 transcription factor

A total of 286 H2O2-sensitive Saccharomyces cerevisiae deletion mutants were screened to identify genes involved in cellular adaptation to H2O2 stress. YAP1, SKN7, GAL11, RPE1, TKL1, IDP1, SLA1, and PET8 were important for adaptation to H2O2. The mutants were divisible into two groups based on their responses to a brief acute dose of H2O2 and to chronic exposure to H2O2. Transcription factors Yap1p, Skn7p, and Gal11p were important for both acute and chronic responses to H2O2. Yap1p and Skn7p were acting in concert for adaptation, which indicates that upregulation of antioxidant functions rather than generation of NADPH or glutathione is important for adaptation. Deletion of GPX3 and YBP1 involved in sensing H2O2 and activating Yap1p affected adaptation but to a lesser extent than YAP1 deletion. NADPH generation was also required for adaptation. RPE1, TKL1, or IDP1 deletants affected in NADPH production were chronically sensitive to H2O2 but resistant to an acute dose, and other mutants affected in NADPH generation tested were similarly affected in adaptation. These mutants overproduced reduced glutathione (GSH) but maintained normal cellular redox homeostasis. This overproduction of GSH was not regulated at transcription of the gene encoding gamma-glutamylcysteine synthetase.

Glutathione regulates the expression of γ‐glutamylcysteine synthetase via the Met4 transcription factor

Our previous studies have shown that glutathione is an essential metabolite in the yeast Saccharomyces cerevisiae because a mutant deleted for GSH1, encoding the first enzyme in γ‐l‐glutamyl‐l‐cysteinylglycine (GSH) biosynthesis, cannot grow in its absence. In contrast, strains deleted for GSH2, encoding the second step in GSH synthesis, grow poorly as the dipeptide intermediate, γ‐glutamylcysteine, can partially substitute for GSH. In this present study, we identify two high copy suppressors that rescue the poor growth of the gsh2 mutant in the absence of GSH. The first contains GSH1, indicating that γ‐glutamylcysteine can functionally replace GSH if it is present in sufficiently high quantities. The second contains CDC34, encoding a ubiquitin conjugating enzyme, indicating a link between the ubiquitin and GSH stress protective systems. We show that CDC34 rescues the growth of the gsh2 mutant by inducing the Met4‐dependent expression of GSH1 and elevating the cellular levels of γ‐glutamylcysteine. Furthermore, this mechanism normally operates to regulate GSH biosynthesis in the cell, as GSH1 promoter activity is induced in a Met4‐dependent manner in a gsh1 mutant which is devoid of GSH, and the addition of exogenous GSH represses GSH1 expression. Analysis of a cis2 mutant, which cannot breakdown GSH, confirmed that GSH and not a metabolic product, serves as the regulatory molecule. However, this is not a general mechanism affecting all Met4‐regulated genes, as MET16 expression is unaffected in a gsh1 mutant, and GSH acts as a poor repressor of MET16 expression compared with methionine. In summary, GSH biosynthesis is regulated in parallel with sulphate assimilation by activity of the Met4 protein, but GSH1‐specific mechanisms exist that respond to GSH availability.

Distinct Redox Regulation in Sub-Cellular Compartments in Response to Various Stress Conditions in Saccharomyces cerevisiae

Responses to many growth and stress conditions are assumed to act via changes to the cellular redox status. However, direct measurement of pH-adjusted redox state during growth and stress has never been carried out. Organellar redox state (E GSH) was measured using the fluorescent probes roGFP2 and pHluorin in Saccharomyces cerevisiae. In particular, we investigated changes in organellar redox state in response to various growth and stress conditions to better understand the relationship between redox-, oxidative- and environmental stress response systems. E GSH values of the cytosol, mitochondrial matrix and peroxisome were determined in exponential and stationary phase in various media. These values (−340 to −350 mV) were more reducing than previously reported. Interestingly, sub-cellular redox state remained unchanged when cells were challenged with stresses previously reported to affect redox homeostasis. Only hydrogen peroxide and heat stress significantly altered organellar redox state. Hydrogen peroxide stress altered the redox state of the glutathione disulfide/glutathione couple (GSSG, 2H+/2GSH) and pH. Recovery from moderate hydrogen peroxide stress was most rapid in the cytosol, followed by the mitochondrial matrix, with the peroxisome the least able to recover. Conversely, the bulk of the redox shift observed during heat stress resulted from alterations in pH and not the GSSG, 2H+/2GSH couple. This study presents the first direct measurement of pH-adjusted redox state in sub-cellular compartments during growth and stress conditions. Redox state is distinctly regulated in organelles and data presented challenge the notion that perturbation of redox state is central in the response to many stress conditions.