Elis C.A. Eleutherio

Acquisition of tolerance against oxidative damage in Saccharomyces cerevisiae

BackgroundLiving cells constantly sense and adapt to redox shifts by the induction of genes whose products act to maintain the cellular redox environment. In the eukaryote Saccharomyces cerevisiae, while stationary cells possess a degree of constitutive resistance towards oxidants, treatment of exponential phase cultures with sub-lethal stresses can lead to the transient induction of protection against subsequent lethal oxidant conditions. The sensors of oxidative stress and the corresponding transcription factors that activate gene expression under these conditions have not yet been completely identified.ResultsWe report the role of SOD1, SOD2 and TPS1 genes (which encode the cytoplasmic Cu/Zn-superoxide dismutase, the mitochondrial Mn-isoform and trehalose-6-phosphate synthase, respectively) in the development of resistance to oxidative stress. In all experimental conditions, the cultures were divided into two parts, one was immediately submitted to severe stress (namely: exposure to H2O2, heat shock or ethanol stress) while the other was initially adapted to 40°C for 60 min. The deficiency in trehalose synthesis did not impair the acquisition of tolerance to H2O2, but this disaccharide played an essential role in tolerance against heat and ethanol stresses. We also verified that the presence of only one Sodp isoform was sufficient to improve cellular resistance to 5 mM H2O2. On the other hand, while the lack of Sod2p caused high cell sensitivity to ethanol and heat shock, the absence of Sod1p seemed to be beneficial to the process of acquisition of tolerance to these adverse conditions. The increase in oxidation-dependent fluorescence of crude extracts of sod1 mutant cells upon incubation at 40°C was approximately 2-fold higher than in sod2 and control strain extracts. Furthermore, in Western blots, we observed that sod mutants showed a different pattern of Hsp104p and Hsp26p expression also different from that in their control strain.ConclusionsTrehalose seemed not to be essential in the acquisition of tolerance to H2O2 stress, but its absence was strongly felt under water stress conditions such as heat and alcoholic stresses. On the other hand, Sod1p could be involved in the control of ROS production; these reactive molecules could signal the induction of genes implicated within cell tolerance to heat and ethanol. The effects of this deletion needs further investigation.

Chaotropicity: a key factor in product tolerance of biofuel-producing microorganisms

Fermentation products can chaotropically disorder macromolecular systems and induce oxidative stress, thus inhibiting biofuel production. Recently, the chaotropic activities of ethanol, butanol and vanillin have been quantified (5.93, 37.4, 174kJ kg(-1)m(-1) respectively). Use of low temperatures and/or stabilizing (kosmotropic) substances, and other approaches, can reduce, neutralize or circumvent product-chaotropicity. However, there may be limits to the alcohol concentrations that cells can tolerate; e.g. for ethanol tolerance in the most robust Saccharomyces cerevisiae strains, these are close to both the solubility limit (<25%, w/v ethanol) and the water-activity limit of the most xerotolerant strains (0.880). Nevertheless, knowledge-based strategies to mitigate or neutralize chaotropicity could lead to major improvements in rates of product formation and yields, and also therefore in the economics of biofuel production.

Antioxidant Protection of Resveratrol and Catechin in Saccharomyces cerevisiae

Moderate consumption of red wine reduces the risk of heart disease and extends lifespan, but the relative contribution of wine polyphenols to these effects is unclear. In this work, the capacity of resveratrol and catechin to protect the eukaryotic microorganism Saccharomyces cerevisiae against oxidative stress caused by different agents, hydrogen peroxide, carbon tetrachloride, and cadmium, was evaluated. Under all stress conditions, both polyphenols increased tolerance, although their protection was more evident under peroxide exposure. By using mutant strains deficient in specific antioxidant defense systems (superoxide dismutases, catalase, or glutathione), it was observed that increased H2O2 tolerance produced by both polyphenols was associated with catalase, as well as the rise in survival rates caused by resveratrol under CCl4. The acquisition of tolerance was correlated with a reduction in lipid peroxidation, indicating that the antioxidant property of resveratrol and catechin involves protection against membrane oxidation.

Revisiting yeast trehalose metabolism.

AbstractEstablishing 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.

Contribution of Yap1 towards Saccharomyces cerevisiae adaptation to arsenic-mediated oxidative stress

In the budding yeast Saccharomyces cerevisiae, arsenic detoxification involves the activation of Yap8, a member of the Yap (yeast AP-1-like) family of transcription factors, which in turn regulates ACR2 and ACR3, genes encoding an arsenate reductase and a plasma-membrane arsenite-efflux protein respectively. In addition, Yap1 is involved in the arsenic adaptation process through regulation of the expression of the vacuolar pump encoded by YCF1 (yeast cadmium factor 1 gene) and also contributing to the regulation of ACR genes. Here we show that Yap1 is also involved in the removal of ROS (reactive oxygen species) generated by arsenic compounds. Data on lipid peroxidation and intracellular oxidation indicate that deletion of YAP1 and YAP8 triggers cellular oxidation mediated by inorganic arsenic. In spite of the increased amounts of As(III) absorbed by the yap8 mutant, the enhanced transcriptional activation of the antioxidant genes such as GSH1 (gamma- glutamylcysteine synthetase gene), SOD1 (superoxide dismutase 1 gene) and TRX2 (thioredoxin 2 gene) may prevent protein oxidation. In contrast, the yap1 mutant exhibits high contents of protein carbonyl groups and the GSSG/GSH ratio is severely disturbed on exposure to arsenic compounds in these cells. These results point to an additional level of Yap1 contribution to arsenic stress responses by preventing oxidative damage in cells exposed to these compounds. Transcriptional profiling revealed that genes of the functional categories related to sulphur and methionine metabolism and to the maintenance of cell redox homoeostasis are activated to mediate adaptation of the wild-type strain to 2 mM arsenate treatment.

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

Abstract 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.

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.

Factors involved with cadmium absorption by a wild-type strain of Saccharomyces cerevisiae

At the concentration used in this work (10 ppm), cadmium was efficiently removed from the environment by stationary yeast cells. While exponential phase cells showed low capacity of cadmium absorption, stationary cells removed 97% of the original metal in 24 hours. Total cadmium absorption shown by dry cells was lower than that of fresh ones, although both cells removed 50% of metal during the first hour of treatment. We also verified that only viable cells were capable of absorbing cadmium. Independently of the growth phase, cells showed high tolerance to 10 ppm CdSO4 and about 80% of cells remained viable after 24 hours exposure to cadmium. However, when stationary phase cells were previously dehydrated and then exposed to cadmium, they exhibited poor survival. By using an oxidation-dependent fluorescent probe, we observed that, once absorbed by cells, cadmium increases the intracellular level of oxidation, which may be responsible for its toxic effect. Crude extracts from stationary phase cells exposed to cadmium showed a 10-fold increase in fluorescence, while extracts from cells of exponential phase did not increase in fluorescence. Dry cells treated with the metal showed a high increase in fluorescence, mainly caused by dehydration.

An iron-based cytosolic catalase and superoxide dismutase mimic complex.

The development of metallodrugs with antioxidant activities is of importance as a way to protect organisms exposed to stressful conditions. Although iron chemistry in the presence of H(2)O(2) is usually associated with pro-oxidant activity, mainly via the Fenton reaction, we found that the mononuclear compound [Fe(HPClNOL)Cl(2)]NO(3) (1; C(15)H(18)Cl(3)FeN(4)O(4), a = 8.7751(3) A, b = 9.0778(4) A, c = 24.3869(10) A, beta = 93.370(2) degrees , monoclinic, P2(1)/c, Z = 4), containing the tripodal ligand 1-[bis(pyridin-2-ylmethyl)amino]-3-chloropropan-2-ol, decomposes hydrogen peroxide and superoxide anion in vitro as well as shows in vivo protection because it prevents the harmful effects promoted by H(2)O(2) on Saccharomyces cerevisiae cells, decreasing the level of lipid peroxidation. This protective effect was observed for wild-type cells, as well as for mutant cells, which do not present the antioxidant metalloenzymes catalase (Ctt1) or copper/zinc superoxide dismutase (Sod1).

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.