Peroxiredoxin promotes longevity and H2O2-resistance in yeast through redox-modulation of protein kinase A.

Peroxiredoxins are H2O2 scavenging enzymes that also carry out H2O2 signaling and chaperone functions. In yeast, the major cytosolic peroxiredoxin, Tsa1 is required for both promoting resistance to H2O2 and extending lifespan upon caloric restriction. We show here that Tsa1 effects both these functions not by scavenging H2O2, but by repressing the nutrient signaling Ras-cAMP-PKA pathway at the level of the protein kinase A (PKA) enzyme. Tsa1 stimulates sulfenylation of cysteines in the PKA catalytic subunit by H2O2 and a significant proportion of the catalytic subunits are glutathionylated on two cysteine residues. Redox modification of the conserved Cys243 inhibits the phosphorylation of a conserved Thr241 in the kinase activation loop and enzyme activity, and preventing Thr241 phosphorylation can overcome the H2O2 sensitivity of Tsa1-deficient cells. Results support a model of aging where nutrient signaling pathways constitute hubs integrating information from multiple aging-related conduits, including a peroxiredoxin-dependent response to H2O2.

Protein Corona Composition of Silica Nanoparticles in Complex Media: Nanoparticle Size does not Matter.

Biomolecules, and particularly proteins, bind on nanoparticle (NP) surfaces to form the so-called protein corona. It is accepted that the corona drives the biological distribution and toxicity of NPs. Here, the corona composition and structure were studied using silica nanoparticles (SiNPs) of different sizes interacting with soluble yeast protein extracts. Adsorption isotherms showed that the amount of adsorbed proteins varied greatly upon NP size with large NPs having more adsorbed proteins per surface unit. The protein corona composition was studied using a large-scale label-free proteomic approach, combined with statistical and regression analyses. Most of the proteins adsorbed on the NPs were the same, regardless of the size of the NPs. To go beyond, the protein physicochemical parameters relevant for the adsorption were studied: electrostatic interactions and disordered regions are the main driving forces for the adsorption on SiNPs but polypeptide sequence length seems to be an important factor as well. This article demonstrates that curvature effects exhibited using model proteins are not determining factors for the corona composition on SiNPs, when dealing with complex biological media.

Glutathione is essential to preserve nuclear function and cell survival under oxidative stress.

Glutathione (GSH) is considered the most important redox buffer of the cell. To better characterize its essential function during oxidative stress conditions, we studied the physiological response of H2O2-treated yeast cells containing various amounts of GSH. We showed that the transcriptional response of GSH-depleted cells is severely impaired, despite an efficient nuclear accumulation of the transcription factor Yap1. Moreover, oxidative stress generates high genome instability in GSH-depleted cells, but does not activate the checkpoint kinase Rad53. Surprisingly, scarce amounts of intracellular GSH are sufficient to preserve cell viability under H2O2 treatment. In these cells, oxidative stress still causes the accumulation of oxidized proteins and the inactivation of the translational activity, but nuclear components and activities are protected against oxidative injury. We conclude that the essential role of GSH is to preserve nuclear function, allowing cell survival and growth resumption after oxidative stress release. We propose that cytosolic proteins are part of a protective machinery that shields the nucleus by scavenging reactive oxygen species before they can cross the nuclear membrane.

Repression of class I transcription by cadmium is mediated by the protein phosphatase 2A.

Toxic metals are part of our environment, and undue exposure to them leads to a variety of pathologies. In response, most organisms adapt their metabolism and have evolved systems to limit this toxicity and to acquire tolerance. Ribosome biosynthesis being central for protein synthesis, we analyzed in yeast the effects of a moderate concentration of cadmium (Cd(2+)) on Pol I transcription that represents >60% of the transcriptional activity of the cells. We show that Cd(2+) rapidly and drastically shuts down the expression of the 35S rRNA. Repression does not result from a poisoning of any of the components of the class I transcriptional machinery by Cd(2+), but rather involves a protein phosphatase 2A (PP2A)-dependent cellular signaling pathway that targets the formation/dissociation of the Pol I-Rrn3 complex. We also show that Pol I transcription is repressed by other toxic metals, such as Ag(+) and Hg(2+), which likewise perturb the Pol I-Rrn3 complex, but through PP2A-independent mechanisms. Taken together, our results point to a central role for the Pol I-Rrn3 complex as molecular switch for regulating Pol I transcription in response to toxic metals.

Impairing the microRNA biogenesis pathway induces proteome modifications characterized by size bias and enrichment in antioxidant proteins.

Perturbation of individual microRNAs, or of the microRNA pathway, plays a role in carcinogenesis. In certain cancer cells, inhibition of the microRNA biogenesis pathway leads to a growth arrest state (CoGAM for Colony Growth Arrest induced by Microprocessor inhibition), which can be rescued by re-expression of individual microRNAs such as miR-20a. We now report that inhibition of the microRNA biogenesis pathway induced proteome changes characterized by a size bias in differentially expressed proteins, with induction of small proteins and inhibition of large ones. This size bias was observed in cells undergoing CoGAM, as well as in CoGAM-resistant cells, and in CoGAM-sensitive cells rescued by miR-20a. In this case, GO analysis of induced proteins identified by mass spectrometry revealed a significant enrichment in proteins involved in resistance to oxidative stress. In addition, H(2) O(2) treatment of Saccharomyces cerevisiae or mammalian cells led to similarly size-biased proteome modifications. Our results point to size bias as a relevant readout of proteome modifications, in particular in conditions of stress such as inhibition of the microRNA biogenesis pathway or oxidative stress. They also suggest research avenues to study the role of the microRNA pathway in proteostasis.

Glutathione degradation is a key determinant of glutathione homeostasis.

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 γ-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 dug2Δ and dug3Δ 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.

Cadmium-glutathione solution structures provide new insights into heavy metal detoxification.

Cadmium is a heavy metal and a pollutant that can be found in large quantities in the environment from industrial waste. Its toxicity for living organisms could arise from its ability to alter thiol-containing cellular components. Glutathione is an abundant tripeptide (γ-Glu-Cys-Gly) that is described as the first line of defence against cadmium in many cell types. NMR experiments for structure and dynamics determination, molecular simulations, competition reactions for metal chelation by different metabolites (γ-Glu-Cys-Gly, α-Glu-Cys-Gly and γ-Glu-Cys) combined with biochemical and genetics experiments have been performed to propose a full description of bio-inorganic reactions occurring in the early steps of cadmium detoxification processes. Our results give unambiguous information about the spontaneous formation, under physiological conditions, of the Cd(GS)(2) complex, about the nature of ligands involved in cadmium chelation by glutathione, and provide insights on the structures of Cd(GS)(2) complexes in solution at different pH. We also show that γ-Glu-Cys, the precursor of glutathione, forms a stable complex with cadmium, but biological studies of the first steps of cadmium detoxification reveal that this complex does not seem to be relevant for this purpose.

Mass spectrometry-based methods for the determination of sulfur and related metabolite concentrations in cell extracts.

The sulfur metabolic pathway plays a central role in cell metabolism. It provides the sulfur amino acids methionine and cysteine, which are essential for protein synthesis, homocysteine, which lies at a critical juncture of this pathway, S-adenosylmethionine, the universal methyl donor in the cell, and glutathione (GSH), which has many crucial functions including protection against oxidative stress and xenobiotics. The intracellular level of these metabolites, which are closely connected with other cellular metabolic pathways, is of major importance for cell physiology and health. Three mass spectrometry-based methods for the determination of sulfur metabolites and also related compounds linked to the glutathione biosynthesis pathway are presented and discussed. The first one enables absolute quantification of these metabolites in cell extracts. It is based on liquid chromatography-electrospray triple quadrupole mass spectrometry coupled to (15)N uniform metabolic labeling of the yeast Saccharomyces cerevisiae. The two other methods are global approaches to metabolite detection involving a high-resolution mass spectrometer, the LTQ-Orbitrap. Ions related to metabolites of interest are picked up from complex and information-rich metabolic fingerprints. By these means, it is possible to detect analytical information outside the initial scope of investigation.

Endoplasmic reticulum is a major target of cadmium toxicity in yeast.

Cadmium (Cd(2+)) is a very toxic metal that causes DNA damage, oxidative stress and apoptosis. Despite many studies, the cellular and molecular mechanisms underlying its high toxicity are not clearly understood. We show here that very low doses of Cd(2+) cause ER stress in Saccharomyces cerevisiae as evidenced by the induction of the unfolded protein response (UPR) and the splicing of HAC1 mRNA. Furthermore, mutant strains (Delta ire1 and Delta hac1) unable to induce the UPR are hypersensitive to Cd(2+), but not to arsenite and mercury. The full functionality of the pathways involved in ER stress response is required for Cd(2+) tolerance. The data also suggest that Cd(2+)-induced ER stress and Cd(2+) toxicity are a direct consequence of Cd(2+) accumulation in the ER. Cd(2+) does not inhibit disulfide bond formation but perturbs calcium metabolism. In particular, Cd(2+) activates the calcium channel Cch1/Mid1, which also contributes to Cd(2+) entry into the cell. The results reinforce the interest of using yeast as a cellular model to study toxicity mechanisms in eukaryotic cells.

Ionizing radiation induces a Yap1-dependent peroxide stress response in yeast.

Repair of DNA damage is fundamental for cellular tolerance to ionizing radiation (IR) and many IR-induced DNA lesions are thought to occur as a result of oxidative stress. We investigated the physiological effects of IR in Saccharomyces cerevisiae by performing protein expression profiles in cells exposed to electron pulse irradiation. Transient induction of several antioxidant enzymes in wild-type cells, but not in cells lacking the oxidative stress regulator Yap1, indicated that IR exposure causes cellular oxidative stress. Yap1 activation involved oxidation to the intramolecular disulfide bond, a signature of activation by peroxide, and was dependent on the Yap1 peroxide sensor Orp1/Gpx3. H(2)O(2) was produced in the culture medium of irradiated cells and was both necessary and sufficient for IR-induced Yap1 activation. When IR was performed in the presence of N(2)O, obviating H(2)O(2) production and increasing hydroxyl radical ((*)OH) production, the Yap1 response was lost, indicating that Yap1 was unable to respond to (*)OH or (*)OH-induced damage. However, the Yap1 response to IR did not seem to be a primary determinant of cellular IR tolerance. Altogether, these data provide a molecular demonstration that cells experience in vivo peroxide stress during IR and indicate that the H(2)O(2) produced cannot account for IR toxicity.

Quantitative transcriptome, proteome, and sulfur metabolite profiling of the Saccharomyces cerevisiae response to arsenite.

Arsenic is ubiquitously present in nature, and various mechanisms have evolved enabling cells to evade toxicity and acquire tolerance. Herein, we explored how Saccharomyces cerevisiae (budding yeast) respond to trivalent arsenic (arsenite) by quantitative transcriptome, proteome, and sulfur metabolite profiling. Arsenite exposure affected transcription of genes encoding functions related to protein biosynthesis, arsenic detoxification, oxidative stress defense, redox maintenance, and proteolytic activity. Importantly, we observed that nearly all components of the sulfate assimilation and glutathione biosynthesis pathways were induced at both gene and protein levels. Kinetic metabolic profiling evidenced a significant increase in the pools of sulfur metabolites as well as elevated cellular glutathione levels. Moreover, the flux in the sulfur assimilation pathway as well as the glutathione synthesis rate strongly increased with a concomitant reduction of sulfur incorporation into proteins. By combining comparative genomics and molecular analyses, we pinpointed transcription factors that mediate the core of the transcriptional response to arsenite. Taken together, our data reveal that arsenite-exposed cells channel a large part of assimilated sulfur into glutathione biosynthesis, and we provide evidence that the transcriptional regulators Yap1p and Met4p control this response in concert.

The S. cerevisiae Yap1 and Yap2 transcription factors share a common cadmium-sensing domain.

Towards elucidating the function of Yap2, which remains unclear, we have taken advantage of the C-terminal homology between Yap1 and Yap2. Swapping domains experiments show that the Yap2 C-terminal domain functionally substitutes for the homologous Yap1 domain in the response to Cd, but not to H2O2. We conclude that specificity determinants of the Cd response are encoded within both Yap1 and Yap2 C-terminus, whereas those required for H2O2 response are only present in the Yap1 C-terminus. Furthermore, our results identify FRM2 as Cd-responsive Yap2 target and indicate a possible role of this protein in regulating a metal stress response.

Liquid chromatography-mass spectrometry and 15N metabolic labeling for quantitative metabolic profiling.

Metabolomics, i.e., the global analysis of cellular metabolites, is becoming a powerful tool for gaining insights into biological functions in the postgenomic context. However, absolute quantitation of endogenous metabolites in biological media remains an issue, and available technologies for the analysis of metabolome still lack robustness and accuracy. We describe here a new method based on liquid chromatography-mass spectrometry and (15)N uniform metabolic labeling of Saccharomyces cerevisiae for accurate and absolute quantitation of nitrogen-containing cell metabolites in metabolic profiling experiments. As a proof of concept study, eight sulfur metabolites involved in the glutathione biosynthesis pathway (i.e., cysteine, homocysteine, methionine, gamma-glutamylcysteine, cystathionine, reduced and oxidized forms of glutathione, and S-adenosylhomocysteine) were simultaneously quantified. The analytical method has been validated by studies of stability, selectivity, precision, and linearity and by the determination of the limits of detection and quantification. It was then applied to the analysis of extracts from cadmium-treated yeasts. In these conditions, the intracellular concentrations of most of the metabolites involved in the glutathione biosynthesis pathway were increased when compared to control extracts. These data correlate with previous proteomic results and also underline the importance of glutathione in cadmium detoxication.

Combined proteome and metabolite-profiling analyses reveal surprising insights into yeast sulfur metabolism.

Metabolomics is considered as an emerging new tool for functional proteomics in the identification of new protein function or in projects aiming at modeling whole cell metabolism. When combined with proteome studies, metabolite-profiling analyses revealed unanticipated insights into the yeast sulfur pathway. In response to cadmium, the observed overproduction of glutathione, essential for the detoxification of the metal, can be entirely accounted for by a marked drop in sulfur-containing protein synthesis and a redirection of sulfur metabolite fluxes to the glutathione pathway. A kinetic analysis showed sequential and dramatic changes in intermediate sulfur metabolite pools within the first hours of the treatment. Strikingly, whereas proteome and metabolic data were positively correlated under cadmium conditions, proteome and metabolic data were negatively correlated during other growth conditions, i.e. methionine supplementation or sulfate starvation. These differences can be explained by alternative mechanisms in the regulation of Met4, the activator of the sulfur pathway. Whereas Met4 activity is controlled by the cellular cysteine content in response to sulfur source and availability, the present study suggests that Met4 activation under cadmium conditions is cysteine-independent. The results clearly indicate that the metabolic state of a cell cannot be safely predicted based solely on proteomic and/or gene expression data. Combined metabolome and proteome studies are necessary to draw a comprehensive and integrated view of cell metabolism.

ATP-dependent reduction of cysteine-sulphinic acid by S. cerevisiae sulphiredoxin.

Proteins contain thiol-bearing cysteine residues that are sensitive to oxidation, and this may interfere with biological function either as 'damage' or in the context of oxidant-dependent signal transduction. Cysteine thiols oxidized to sulphenic acid are generally unstable, either forming a disulphide with a nearby thiol or being further oxidized to a stable sulphinic acid. Cysteine-sulphenic acids and disulphides are known to be reduced by glutathione or thioredoxin in biological systems, but cysteine-sulphinic acid derivatives have been viewed as irreversible protein modifications. Here we identify a yeast protein of relative molecular mass M(r) = 13,000, which we have named sulphiredoxin (identified by the US spelling 'sulfiredoxin', in the Saccharomyces Genome Database), that is conserved in higher eukaryotes and reduces cysteine-sulphinic acid in the yeast peroxiredoxin Tsa1. Peroxiredoxins are ubiquitous thiol-containing antioxidants that reduce hydroperoxides and control hydroperoxide-mediated signalling in mammals. The reduction reaction catalysed by sulphiredoxin requires ATP hydrolysis and magnesium, involving a conserved active-site cysteine residue which forms a transient disulphide linkage with Tsa1. We propose that reduction of cysteine-sulphinic acids by sulphiredoxin involves activation by phosphorylation followed by a thiol-mediated reduction step. Sulphiredoxin is important for the antioxidant function of peroxiredoxins, and is likely to be involved in the repair of proteins containing cysteine-sulphinic acid modifications, and in signalling pathways involving protein oxidation.

The control of the yeast H2O2 response by the Msn2/4 transcription factors.

We have analysed the contribution of the Msn2/4 transcription factors and the Ras-cAMP-protein kinase A (PKA) pathway to the control of the yeast H2O2 response. Strains deleted for MSN2 and MSN4 are hypersensitive to H2O2, although they can still adapt to this oxidant. They are also unable to induce 27 proteins of the H2O2 stimulon as shown by quantitative two-dimensional gel analysis. This peculiar H2O2 tolerance defect, the nature of the proteins of the Msn2/4 regulon, and the partial overlap of this regulon with the Yap1 H2O2-response regulon, suggest an independent and distinctive role of these two H2O2 stress response pathways. A strain lacking PDE2, and therefore carrying high intracellular cAMP levels, is also hypersensitive to H2O2. In the presence of exogenous cAMP, this strain does not induce the entire H2O2 Msn2/4 regulon and some other proteins. This, and the normal H2O2 induction of a gene reporter under control of the Yap1 regulator when intracellular cAMP level are high, demonstrate that the Ras-cAMP pathway negatively affects the H2O2 stress response through Msn2/4. However, the high H2O2 sensitivity of a strain lacking the PKA-negative regulatory subunit Bcy1, is not only the consequence of the inhibition of Msn2/4 but also of Yap1 through a yet undefined mechanism.

Sulfur sparing in the yeast proteome in response to sulfur demand.

Genome-wide studies have recently revealed the unexpected complexity of the genetic response to apparently simple physiological changes. Here, we show that when yeast cells are exposed to Cd(2+), most of the sulfur assimilated by the cells is converted into glutathione, a thiol-metabolite essential for detoxification. Cells adapt to this vital metabolite requirement by modifying globally their proteome to reduce the production of abundant sulfur-rich proteins. In particular, some abundant glycolytic enzymes are replaced by sulfur-depleted isozymes. This global change in protein expression allows an overall sulfur amino acid saving of 30%. This proteomic adaptation is essentially regulated at the mRNA level. The main transcriptional activator of the sulfate assimilation pathway, Met4p, plays an essential role in this sulfur-sparing response.

Involvement of superoxide dismutases in the response of Escherichia coli to selenium oxides.

Selenium can provoke contrasting effects on living organisms. It is an essential trace element, and low concentrations have beneficial effects, such as the reduction of the incidence of cancer. However, higher concentrations of selenium salts can be toxic and mutagenic. The bases for both toxicity and protection are not clearly understood. To provide insights into these mechanisms, we analyzed the proteomic response of Escherichia coli cells to selenate and selenite treatment under aerobic conditions. We identified 23 proteins induced by both oxides and ca. 20 proteins specifically induced by each oxide. A striking result was the selenite induction of 8 enzymes with antioxidant properties, particularly the manganese and iron superoxide dismutases (SodA and SodB). The selenium inductions of sodA and sodB were controlled by the transcriptional regulators SoxRS and Fur, respectively. Strains with decreased superoxide dismutase activities were severely impaired in selenium oxide tolerance. Pretreatment with a sublethal selenite concentration triggered an adaptive response dependent upon SoxRS, conferring increased selenite tolerance. Altogether, our data indicate that superoxide dismutase activity is essential for the cellular defense against selenium salts, suggesting that superoxide production is a major mechanism of selenium toxicity under aerobic conditions.

Identification in Saccharomyces cerevisiae of a new stable variant of alkyl hydroperoxide reductase 1 (Ahp1) induced by oxidative stress.

Yeasts lacking cytoplasmic superoxide dismutase (Cu,Zn-SOD) activity are permanently subjected to oxidative stress. We used two-dimensional PAGE to examine the proteome pattern of Saccharomyces cerevisiae strains lacking Cu,Zn-SOD. We found a new stable form of alkyl hydroperoxide reductase 1 (Ahp1) with a lower isoelectric point. This form was also present in wild type strains after treatment with tert-butyl hydroperoxide. In vitro enzyme assays showed that Ahp1p had lower specific activity in strains lacking Cu,Zn-SOD. We studied three mutants presenting a reduced production of the low pI variant under oxidative stress conditions. Two of the mutants (C62S and S59D) were totally inactive, thus suggesting that the acidic form of Ahp1p may only appear when the enzyme is functional. The other mutant (S59A) was active in vitro and was more resistant to inactivation by tert-butyl hydroperoxide than the wild type enzyme. Furthermore, the inactivation of Ahp1p in vitro is correlated with its conversion to the low pI form. These results suggest that in vivo during some particular oxidative stress (alkyl hydroperoxide treatment or lack of Cu,Zn-SOD activity but not hydrogen peroxide treatment), the catalytic cysteine of Ahp1p is more oxidized than cysteine-sulfenic acid (a natural occurring intermediate of the enzymatic reaction) and that cysteine-sulfinic acid or cysteine-sulfonic acid variant may be inactive.