Daughters of the budding yeast from old mothers have shorter replicative lifespans but not total lifespans. Are DNA damage and rDNA instability the factors that determine longevity?

Although a lot of effort has been put into the search for factors responsible for aging in yeast mother cells, our knowledge of cellular changes in daughter cells originating from old mothers is still very limited. It has been shown that an old mother is not able to compensate for all negative changes within its cell and therefore transfers them to the bud. In this paper, we show for the first time that daughter cells of an old mother have a reset lifespan expressed in units of time despite drastic reduction of their budding lifespan, which suggests that a single yeast cell has a fixed programmed longevity regardless of the time point at which it was originated. Moreover, in our study we found that longevity parameters are not correlated with the rDNA level, DNA damage, chromosome structure or aging parameters (budding lifespan and total lifespan).

Copy number variations of genes involved in stress responses reflect the redox state and DNA damage in brewing yeasts.

The yeast strains of the Saccharomyces sensu stricto complex involved in beer production are a heterogeneous group whose genetic and genomic features are not adequately determined. Thus, the aim of the present study was to provide a genetic characterization of selected group of commercially available brewing yeasts both ale top-fermenting and lager bottom-fermenting strains. Molecular karyotyping revealed that the diversity of chromosome patterns and four strains with the most accented genetic variabilities were selected and subjected to genome-wide array-based comparative genomic hybridization (array-CGH) analysis. The differences in the gene copy number were found in five functional gene categories: (1) maltose metabolism and transport, (2) response to toxin, (3) siderophore transport, (4) cellular aldehyde metabolic process, and (5) L-iditol 2-dehydrogenase activity (p < 0.05). In the Saflager W-34/70 strain (Fermentis) with the most affected array-CGH profile, loss of aryl-alcohol dehydrogenase (AAD) gene dosage correlated with an imbalanced redox state, oxidative DNA damage and breaks, lower levels of nucleolar proteins Nop1 and Fob1, and diminished tolerance to fermentation-associated stress stimuli compared to other strains. We suggest that compromised stress response may not only promote oxidant-based changes in the nucleolus state that may affect fermentation performance but also provide novel directions for future strain improvement.

Affected chromosome homeostasis and genomic instability of clonal yeast cultures.

Yeast cells originating from one single colony are considered genotypically and phenotypically identical. However, taking into account the cellular heterogeneity, it seems also important to monitor cell-to-cell variations within a clone population. In the present study, a comprehensive yeast karyotype screening was conducted using single chromosome comet assay. Chromosome-dependent and mutation-dependent changes in DNA (DNA with breaks or with abnormal replication intermediates) were studied using both single-gene deletion haploid mutants (bub1, bub2, mad1, tel1, rad1 and tor1) and diploid cells lacking one active gene of interest, namely BUB1/bub1, BUB2/bub2, MAD1/mad1, TEL1/tel1, RAD1/rad1 and TOR1/tor1 involved in the control of cell cycle progression, DNA repair and the regulation of longevity. Increased chromosome fragility and replication stress-mediated chromosome abnormalities were correlated with elevated incidence of genomic instability, namely aneuploid events-disomies, monosomies and to a lesser extent trisomies as judged by in situ comparative genomic hybridization (CGH). The tor1 longevity mutant with relatively balanced chromosome homeostasis was found the most genomically stable among analyzed mutants. During clonal yeast culture, spontaneously formed abnormal chromosome structures may stimulate changes in the ploidy state and, in turn, promote genomic heterogeneity. These alterations may be more accented in selected mutated genetic backgrounds, namely in yeast cells deficient in proper cell cycle regulation and DNA repair.

Shifts in rDNA levels act as a genome buffer promoting chromosome homeostasis.

The nucleolus is considered to be a stress sensor and rDNA-based regulation of cellular senescence and longevity has been proposed. However, the role of rDNA in the maintenance of genome integrity has not been investigated in detail. Using genomically diverse industrial yeasts as a model and array-based comparative genomic hybridization (aCGH), we show that chromosome level may be balanced during passages and as a response to alcohol stress that may be associated with changes in rDNA pools. Generation- and ethanol-mediated changes in genes responsible for protein and DNA/RNA metabolism were revealed using next-generation sequencing. Links between redox homeostasis, DNA stability, and telomere and nucleolus states were also established. These results suggest that yeast genome is dynamic and chromosome homeostasis may be controlled by rDNA.

Genetic profiling of yeast industrial strains using in situ comparative genomic hybridization (CGH).

The genetic differences and changes in genomic stability may affect fermentation processes involving baker's, brewer's and wine yeast strains. Thus, it seems worthwhile to monitor the changes in genomic DNA copy number of industrial strains. In the present study, we developed an in situ comparative genomic hybridization (CGH) to investigate the ploidy and genetic differences between selected industrial yeast strains. The CGH-based system was validated using the laboratory Saccharomyces cerevisiae yeast strains (haploid BY4741 and diploid BY4743). DNA isolated from BY4743 cells was considered a reference DNA. The ploidy and DNA gains and losses of baker's, brewer's and wine strains were revealed. Taken together, the in situ CGH was shown a helpful molecular tool to identify genomic differences between yeast industrial strains. Moreover, the in situ CGH-based system may be used at the single-cell level of analysis to supplement array-based techniques and high-throughput analyses at the population scale.

Single-cell analysis of aneuploidy events using yeast whole chromosome painting probes (WCPPs).

Aneuploidy is considered a widespread genetic variation in such cell populations as yeast strains, cell lines and cancer cells, and spontaneous changes in the chromosomal copy number may have implications for data interpretation. Thus, aneuploidy monitoring is essential during routine laboratory practice, especially while conducting biochemical and/or gene expression analyses. In the present study, we constructed a panel of whole chromosome painting probes (WCPPs) to monitor aneuploidy in a single yeast Saccharomyces cerevisiae cell. The WCPP-based system was validated using "normal" haploid and diploid cells, as well as disomic cells both with and without cell synchronisation. FISH that utilised WCPPs was combined with DNA cell cycle analysis (imaging cytometry) to provide a detailed analysis of signal variability during the cell cycle. Chromosome painting can be utilised to detect spontaneously formed disomic chromosomes and study aneuploidy-promoting conditions. For example, the frequency of disomic chromosomes was increased in cells lacking NAD(+)-dependent histone deacetylase Sir2p compared with wild-type cells (p<0.05). In conclusion, WCPPs may be considered to be a powerful molecular tool to identify individual genomic differences. Moreover, the WCPP-based system may be used at the single-cell level of analysis to supplement array-based techniques and high-throughput analyses at the population scale.

Revisiting yeast trehalose metabolism.

Establishing the function of trehalose in yeast cells has led us, over the years, through a long path-from simple energy storage carbohydrate, then a stabilizer and protector of membranes and proteins, through a safety valve against damage caused by oxygen radicals, up to regulator of the glycolytic path. In addition, trehalose biosynthesis has been proposed as a target for novel drugs against several pathogens. Since this pathway is entirely absent in mammalian cells and makes use of highly specific enzymes, trehalose metabolism might be an interesting target for the development of novel therapies. In this review, we want to address some recent points investigated about trehalose metabolism in Saccharomyces cerevisiae, focusing mainly on the mechanism by which this simple disaccharide protects against stress and on the enzymes involved in its synthesis and breakdown. We believe that these concepts are of great importance for medical and biotechnological applications.

Regulation of the yeast trehalose-synthase complex by cyclic AMP-dependent phosphorylation.

BACKGROUND: Trehalose is an important protectant in several microorganisms. In Saccharomyces cerevisiae, it is synthesized by a large complex comprising the enzymes Tps1 and Tps2 and the subunits Tps3 and Tsl1, showing an intricate metabolic control. METHODS: To investigate how the trehalose biosynthesis pathway is regulated, we analyzed Tps1 and Tps2 activities as well as trehalose and trehalose-6-phosphate (T6P) contents by mass spectrometry. RESULTS: Tsl1 deficiency totally abolished the increase in Tps1 activity and accumulation of trehalose in response to a heat stress, whereas absence of Tps3 only reduced Tps1 activity and trehalose synthesis. In extracts of heat stressed cells, Tps1 was inhibited by T6P and by ATP. Mg(2+) in the presence of cAMP. In contrast, cAMP-dependent phosphorylation did not inhibit Tps1 in tps3 cells, which accumulated a higher proportion of T6P after stress. Tps2 activity was not induced in a tps3 mutant. CONCLUSION: Taken together these results suggest that Tsl1 is a decisive subunit for activity of the TPS complex since in its absence no trehalose synthesis occurred. On the other hand, Tps3 seems to be an activator of Tps2. To perform this task, Tps3 must be non-phosphorylated. To readily stop trehalose synthesis during stress recovery, Tps3 must be phosphorylated by cAMP-dependent protein kinase, decreasing Tps2 activity and, consequently, increasing the concentration of T6P which would inhibit Tps1. GENERAL SIGNIFICANCE: A better understanding of TPS complex regulation is essential for understanding how yeast deals with stress situations and how it is able to recover when the stress is over.

The involvement of GSH in the activation of human Sod1 linked to FALS in chronologically aged yeast cells.

Mutations in Cu, Zn-superoxide dismutase (Sod1) have been associated with familial amyotrophic lateral sclerosis, an age-related disease. Because several studies suggest that oxidative stress plays a central role in neurodegeneration, we aimed to investigate the role of the antioxidant glutathione (GSH) in the activation of human A4V Sod1 during chronological aging. Transformation of wild-type and A4V hSod1 into a gsh null mutant and in its parental strain of Saccharomyces cerevisiae indicated that during aging, the number of viable cells was strongly influenced by A4V hSod1 mainly in cells lacking GSH. Activity of hSod1 increased in response to aging, although the increase observed in A4V hSod1 was almost 60% lower. Activation of hSod1 (A4V and WT) did not occur after aging, in cells lacking GSH, but could still be observed in the absence of Ccs1. Furthermore, no increase in activity could be seen in grx1 and grx2 null mutants, suggesting that glutathionylation is essential for hSod1 activation. The A4V mutation as well as the absence of GSH, reduced hSod1 activity, and increased oxidative damage after aging. In conclusion, our results point to a GSH requirement for hSod1 Ccs1-independent activation as well as for protection of hSod1 during the aging process.

The effect of trehalose on the fermentation performance of aged cells of Saccharomyces cerevisiae.

The fermentation process offers a wide variety of stressors for yeast, such as temperature, aging, and ethanol. To evaluate a possible beneficial effect of trehalose on ethanol production, we used mutant strains of Saccharomyces cerevisiae possessing different deficiencies in the metabolism of this disaccharide: in synthesis, tps1; in transport, agt1; and in degradation, ath1 and nth1. According to our results, the tps1 mutant, the only strain tested unable to synthesize trehalose, showed the lowest fermentation yield, indicating that this sugar is important to improve ethanol production. At the end of the first fermentation cycle, only the strains deficient in transport and degradation maintained a significant level of the initial trehalose. The agt1, ath1, and nth1 strains showed the highest survival rates and the highest proportions of non-petites. Accumulation of petites during fermentation has been correlated to low ethanol production. When recycled back for a subsequent fermentation, those mutant strains produced the highest ethanol yields, suggesting that trehalose is required for improving fermentative capacity and longevity of yeasts, as well as their ability to withstand stressful industrial conditions. Finally, according to our results, the mechanism by which trehalose improves ethanol production seems to involve mainly protection against protein oxidation.

Cytotoxicity mechanism of two naphthoquinones (menadione and plumbagin) in Saccharomyces cerevisiae.

BACKGROUND: Quinones are compounds extensively used in studies of oxidative stress due to their role in plants as chemicals for defense. These compounds are of great interest for pharmacologists and scientists, in general, because several cancer chemotherapeutic agents contain the quinone nucleus. However, due to differences in structures and diverse pharmacological effects, the exact toxicity mechanisms exerted by quinones are far from elucidatation. METHODOLOGY/PRINCIPAL FINDINGS: Using Saccharomyces cerevisiae, we evaluated the main mechanisms of toxicity of two naphthoquinones, menadione and plumbagin, by determining tolerance and oxidative stress biomarkers such as GSH and GSSG, lipid peroxidation levels, as well as aconitase activity. The importance of glutathione transferases (GST) in quinone detoxification was also addressed. The GSSG/GSH ratio showed that menadione seemed to exert its toxicity mainly through the generation of ROS while plumbagin acted as an electrophile reacting with GSH. However, the results showed that, even by different pathways, both drugs were capable of generating oxidative stress through their toxic effects. Our results showed that the control strain, BY4741, and the glutathione transferase deficient strains (gtt1Delta and gtt2Delta) were sensitive to both compounds. With respect to the role of GST isoforms in cellular protection against quinone toxicity, we observed that the Gtt2 deficient strain was unable to overcome lipid peroxidation, even after a plumbagin pre-treatment, indicating that this treatment did not improve tolerance when compared with the wild type strain. Cross-tolerance experiments confirmed distinct cytotoxicity mechanisms for these naphthoquinones since only a pre-treatment with menadione was able to induce acquisition of tolerance against stress with plumbagin. CONCLUSIONS/SIGNIFICANCE: These results suggest different responses to menadione and plumbagin which could be due to the fact that these compounds use different mechanisms to exert their toxicity. In addition, the Gtt2 isoform seemed to act as a general protective factor involved in quinone detoxification.

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

Glutathione transferases are detoxifying enzymes responsible for eliminating toxic compounds generated under a variety of stress conditions. Saccharomyces cerevisiae control cells and glutathione transferase mutant strains (gtt1 and gtt2) were used to analyze tolerance, lipid and protein oxidation as oxidative stress markers during growth in the presence of H2O2. Glucose 6-phosphate dehydrogenase (G6PD) and glutathione reductase were assayed to monitor the capacity of cells to recycle glutathione. Although a reduction in growth was observed, deletion of GTT1 showed less inhibition by H2O2 than the control strain. Cells showed a significant reduction in cellular viability during the first hours of growth, the gtt1 mutant being hypersensitive even after 24 h of H2O2 exposure. As a consequence of oxidative stress caused by exposure to H2O2, an increase in lipid peroxidation was observed, mainly in the glutathione transferase mutant strains. While protein carbonylation increased by 17% and 23%, respectively, after 2 h in the presence of H2O2 in the control and gtt2 mutant, a 40% increase was observed in the gtt1 strain after 24-h exposure. The antioxidant G6PD and glutathione reductase activities were affected in the gtt1 mutant during H2O2 exposure, which could be critical for recycling glutathione. The same was observed for the gtt2 mutant after 2-h treatment, indicating that glutathione recycling might be associated with the detoxification process. Thus, glutathione transferases, Gtt1 and Gtt2, seem to be crucial in the response to H2O2 stress.

Glutathione is necessary to ensure benefits of calorie restriction during ageing in Saccharomyces cerevisiae.

Calorie restriction increases longevity of mammals and yeasts but this mechanism remains unclear. In this study, the role of glutathione on lifespan extension induced by calorie restriction was investigated by using a Saccharomyces cerevisiae strain deficient in glutathione synthesis (gsh1). We observed an increase in chronological lifespan of calorie-restricted gsh1 mutant cells, compared to WT (wild type) strain, which was associated with a reduction in the levels of oxidative stress biomarkers. The gsh1 strain showed an increase in cell yield under calorie restriction that was associated with a higher pyruvate kinase activity and a reduction in oxygen consumption and aconitase activity. This indicates that the respiratory metabolism is decreased in gsh1 mutant cells. The lifespan extension of gsh1 mutant cells did not represent an advantage at long term, since old cells of gsh1 strain showed a higher frequency of petite mutants. In addition, aged WT cells outlast aged gsh1 mutant cells in direct competition assays in a fresh medium. These results suggest that glutathione is required for the beneficial effects of calorie restriction on cellular longevity.

The role of trehalose and its transporter in protection against reactive oxygen species.

During menadione stress, trehalose was necessary intracellularly, but under H2O2, the sugar was required on the outside of the plasma membrane. The mechanism of protection involves minimizing the oxidative damage caused to both proteins and lipids, which would require the presence of trehalose on both sides of the lipid bilayer.

Apoptosis as a mechanism for removal of mutated cells of Saccharomyces cerevisiae: the role of Grx2 under cadmium exposure.

Cadmium is a strong mutagen that acts by inhibiting DNA mismatch repair, while its toxic effect seems to be related to an indirect oxidative stress that involves glutathione (GSH) mobilization. Among the roles of GSH is the protection of proteins against oxidative damage, by forming reversible mixed disulfides with cysteine residues, a process known as protein glutathionylation and catalyzed by glutaredoxins (Grx). In this current study, Saccharomyces cerevisiae cells deficient in GRX2, growing in 80 muM CdSO(4), showed high mitochondrial mutagenic rate, determined by frequency of mutants that had lost mitochondrial function (petite mutants), high tolerance and lower apoptosis induction. The mutant strain also showed decreased levels of glutathionylated-protein after cadmium exposure, which might difficult the signaling to apoptosis, leading to increased mutagenic rates. Taken together, these results suggest that Grx2 is involved with the apoptotic death induced by cadmium, a form of cellular suicide that might lead of removal of mutated cells.

Oxidative stress response in eukaryotes: effect of glutathione, superoxide dismutase and catalase on adaptation to peroxide and menadione stresses in Saccharomyces cerevisiae.

Aiming to clarify the mechanisms by which eukaryotes acquire tolerance to oxidative stress, adaptive and cross-protection responses to oxidants were investigated in Saccharomyces cerevisiae. Cells treated with sub-lethal concentrations of menadione (a source of superoxide anions) exhibited cross-protection against lethal doses of peroxide; however, cells treated with H2O2 did not acquire tolerance to a menadione stress, indicating that menadione response encompasses H2O2 adaptation. Although, deficiency in cytoplasmic superoxide dismutase (Sod1) had not interfered with response to superoxide, cells deficient in glutathione (GSH) synthesis were not able to acquire tolerance to H2O2 when pretreated with menadione. These results suggest that GSH is an inducible part of the superoxide adaptive stress response, which correlates with a decrease in the levels of intracellular oxidation. On the other hand, neither the deficiency of Sod1 nor in GSH impaired the process of acquisition of tolerance to H2O2 achieved by a mild pretreatment with peroxide. Using a strain deficient in the cytosolic catalase, we were able to conclude that the reduction in lipid peroxidation levels produced by the adaptive treatment with H2O2 was dependent on this enzyme. Corroborating these results, the pretreatment with low concentrations of H2O2 promoted an increase in catalase activity.

Vacuolar compartmentation of the cadmium-glutathione complex protects Saccharomyces cerevisiae from mutagenesis.

In the yeast Saccharomyces cerevisiae, gamma-glutamyl transferase (gamma-GT; EC 2.3.2.2) is a vacuolar-membrane bound enzyme. In this work we verified that S. cerevisiae cells deficient in gamma-GT absorbed almost 2.5-fold as much cadmium as the wild-type (wt) cells, suggesting that this enzyme might be responsible for the recycle of cadmium-glutathione complex stored in the vacuole. The mutant strain showed difficulty in keeping constant levels of glutathione (GSH) during the stress, although the GSH-reductase activity was practically the same in both wt and mutant strains, before and after metal stress. This difficulty to maintain the GSH levels in the gamma-GT mutant strain led to high levels of lipid peroxidation and carbonyl proteins in response to cadmium, higher than in the wt, but lower than in a mutant deficient in GSH synthesis. Although the increased levels of oxidative stress, gamma-GT mutant strain showed to be tolerant to cadmium and showed similar mutation rates to the wt, indicating that the compartmentation of the GSH-cadmium complex in vacuole protects cells against the mutagenic action of the metal. Confirming this hypothesis, a mutant strain deficient in Ycf1, which present high concentrations of GSH-cadmium in cytoplasm due to its deficiency in transport the complex to vacuole, showed increased mutation rates.

The role of cytoplasmic catalase in dehydration tolerance of Saccharomyces cerevisiae.

In this study, we investigated the role played by cytoplasmic catalase (Ctt1) in resistance against water loss using the yeast Saccharomyces cerevisiae as eukaryotic cell model. Comparing a mutant possessing a specific lesion in CTT1 with its parental strain, it was observed that both control and ctt1 strains exhibited increased levels of lipid peroxidation after dehydration, suggesting that catalase does not protect membranes during drying. Although the ctt1 strain has only 1 catalase isoform (peroxisomal catalase), the mutant showed the same levels of total catalase activity as the control strain. Furthermore, in cells deficient in Ctt1, the reduced glutathione:oxidized glutathione ratio (GSH:GSSG) of dry cells was higher than that of the control strain, indicating a compensatory mechanism of defense in response to dehydration. Even so, desiccation tolerance of the ctt1 strain was significantly lower than in the control strain. Using a fluorescent probe sensitive to oxidation, we observed that cells of the ctt1 strain showed levels of intracellular oxidation 70% higher than those of control strain, suggesting that Ctt1 plays a role in the maintenance of the intracellular redox balance during dehydration and, therefore, in tolerance against a water stress.

The role of glutathione transferases in cadmium stress.

Using Saccharomyces cerevisiae as experimental model, we observed that cells mutated in the GTT1 or GTT2 genes showed twice as much cadmium absorption than the control strain. We proposed that the formation of the cadmium-glutathione complex is dependent on that transferase, since it was previously demonstrated that the cytoplasmic levels of this complex affect cadmium uptake. The addition of glutathione monoethyl ester (GME), a drug that mimics glutathione (GSH), to gtt1Delta cells restored the levels of metal absorption to those of the control strain. However, with respect to gtt2Delta cells, addition of GME did not alter the capacity of removing cadmium from the medium. Taken together, these results suggest that Gtt1 and Gtt2 play different roles in the mechanism of cadmium detoxification. By analyzing the toxic effect of this metal, we verified that gtt2Delta and gsh1Delta cells showed, respectively, higher and lower tolerance to cadmium stress than control cells, suggesting that although GSH plays a relevant role in cell protection, formation of the GSH-Cd conjugate is deleterious to the mechanism of defense.

The effect of superoxide dismutase deficiency on cadmium stress.

Saccharomyces cerevisiae mutant strains deficient in superoxide dismutase (Sod), an antioxidant enzyme, were used to analyze cadmium absorption and the oxidation produced by it. Cells lacking the cytosolic Sod1 removed twice as much cadmium as the control strain, while those deficient in the mitochondrial Sod2 exhibited poor metal absorption. Interestingly, the sod1 mutant did not become more oxidized after exposure to cadmium, as opposed to the control strain. We observed that the deficiency of Sod1 increases the expression of both Cup1 (a metallothionein) and Ycf1 (a vacuolar glutathione S-conjugate pump), proteins involved with protection against cadmium. Furthermore, when sod1 cells were exposed to cadmium, the ratio glutathione oxidized/glutathione reduced did not increase as expected. We propose that a high level of metallothionein expression would relieve glutathione under cadmium stress, while an increased level of Ycf1 expression would favor compartmentalization of this metal into the vacuole. Both conditions would reduce the level of glutathione-cadmium complex in cytosol, contributing to the high capacity of absorbing cadmium by the sod1 strain. Previous results showed that the glutathione-cadmium complex regulates cadmium uptake. These results indicate that, even indirectly, metallothionein also regulates cadmium transport.