- Email: [email protected]
Glutathione is necessary to ensure benefits of calorie restriction during ageing in Saccharomyces cerevisiae
Mechanisms of Ageing and Development 129 (2008) 700–705
Contents lists available at ScienceDirect
Mechanisms of Ageing and Development journal homepage: www.elsevier.com/locate/mechagedev
Glutathione is necessary to ensure benefits of calorie restriction during ageing in Saccharomyces cerevisiae Se´rgio C. Mannarino a,*, Maria A. Amorim b,c, Marcos D. Pereira a, Pedro Moradas-Ferreira b,c, Anita D. Panek a, Vı´tor Costa b,c, Elis C.A. Eleutherio a a b c
Departamento de Bioquı´mica, Instituto de Quı´mica, UFRJ, 21941-909 Rio de Janeiro, RJ, Brazil IBMC, Rua do Campo Alegre 823, 4150-180 Porto, Portugal ICBAS, Departamento de Biologia Molecular, Universidade do Porto, Porto, Portugal
A R T I C L E I N F O
A B S T R A C T
Article history: Received 28 April 2008 Received in revised form 11 August 2008 Accepted 8 September 2008 Available online 18 September 2008
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. ß 2008 Elsevier Ireland Ltd. All rights reserved.
Keywords: Saccharomyces cerevisiae Chronological ageing Glutathione Calorie restriction
1. Introduction Low-calorie diets known as calorie restriction (CR) extend the lifespan of organisms ranging from yeast to mammals. The molecular mechanisms by which CR slows ageing have been associated with the activation of Sir2-family proteins, modulation of nutrient signaling pathways, decreased production of reactive oxygen species (ROS) and accumulation of oxidative damages (Sohal and Weindruch, 1996; Barja, 2002; Sohal, 2002; Merry, 2004). The budding yeast Saccharomyces cerevisiae has been widely used as a model system to study the mechanisms of modulation of two different types of lifespan: replicative lifespan, measured as the number of daughter cells produced by a mother cell before senescence and chronological lifespan, the length of time a yeast cell can survive in a nondividing state (Jazwinski, 1999; Fabrizio and Longo, 2003; Bitterman et al., 2003; Piper, 2006). In both ageing models, nutrient signaling has been linked to longevity (Kaeberlein et al., 2007). In the lab, yeast cells are typically grown in media containing high levels of glucose (2%) and amino acids. However, independent studies have shown that
* Corresponding author. Tel.: +55 21 2562 7735; fax: +55 21 2562 7735. E-mail address: [email protected] (S.C. Mannarino). 0047-6374/$ – see front matter ß 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mad.2008.09.001
replicative and chronological lifespan can be increased by reducing either glucose or amino acid concentration (or both) (ReverterBranchat et al., 2004; Fabrizio et al., 2003, 2005; Jiang et al., 2000; Lin et al., 2000; Powers et al., 2006). A reduction in glucose concentration results in a metabolic shift from fermentation to respiration (Lin et al., 2002). While it is often incorrectly assumed that respiration and ROS production are directly proportional, the biological consequences of changes in respiration on ROS production and longevity are complex and far from being completely understood (Balaban et al., 2005; Speakman et al., 2004). Lifespan extension has been associated to both increase (Barros et al., 2004; Speakman et al., 2004; Bonawitz et al., 2006) and decrease (Feng et al., 2001; Dillin et al., 2002) in respiration rate. In yeast, Barros et al. (2004) suggested that CR increases mitochondrial respiration but decreases the ratio (ROS released)/ (O2 consumed) by reducing the leakage of electrons from the respiratory chain. A more recent study shows, however, that the partial uncoupling of oxidative phosphorylation in human fibroblasts and yeast mother cells increases ROS production and induces premature senescence (Sto¨ckl et al., 2007). The central regulatory role of mitochondrial metabolism in both replicative and chronological ageing in yeast is further supported by different studies. Yeast cells pre-grown on a respiratory carbon source, compared to glucose grown cells, have a higher chron-
S.C. Mannarino et al. / Mechanisms of Ageing and Development 129 (2008) 700–705
ological longevity. This prior adaptation to efficient respiratory maintenance also ensures that such chronological aged cells still display a full replicative potential should they reenter the cell cycle (Piper et al., 2006). Indeed, yeast longevity is extended by overexpression of the HAP4 transcription factor, which increases the expression of numerous respiratory genes (Lin et al., 2002; Piper et al., 2006), or overexpression of mitochondrial NADdependent dehydrogenases (Lin et al., 2004). Mitochondrial dysfunction associated with ageing increases ROS production leading to the accumulation of oxidative damages to proteins, lipids and DNA. In agreement, studies in yeast and other model organisms showed that lifespan is shortened in antioxidant deficient cells and the overexpression of antioxidant defenses delays ageing (reviewed by Piper, 2006). Some studies have suggested that CR functions to extend lifespan by increasing resistance to ROS (Sohal and Weindruch, 1996). Glutathione (gamma-glutamylcysteinylglycine; GSH) is a low-molecular-mass thiol with proposed functions in many cellular processes including protection against xenobiotics, carcinogens and ROS, being used as a co-factor for glutathione peroxidase or protecting cysteine residues in proteins through S-thiolation (Thomas and Mallis, 2001). The biological importance of GSH is dependent upon the redox-active free sulfydryl moiety of its cysteine residue. In this work, we investigated the importance of the antioxidant metabolite glutathione to ensure full benefits of CR in cellular longevity. We observed that yeast cells deficient in glutathione production (gsh1 strain) have an increased chronological lifespan, compared to wild-type cells, and present lower levels of oxidative stress markers. We noted a difference between both strains regarding their energy metabolism, where respiration rate is higher in the gsh1 mutant strain. However, aged gsh1 mutant cells show a higher mitochondrial mutagenic rate and are not able to compete with aged wild type when they are in the same environment. 2. Materials and methods 2.1. Yeast strains and growth conditions Saccharomyces cerevisiae wild-type strain BY4741 (MATa; his3; leu2; met15; ura3) and its isogenic mutant gsh1 harboring the gene GSH1 interrupted by the gene KanMX4, were acquired from Euroscarf, Frankfurt, Germany. Stocks of yeast strains were maintained on solid YPD 2% medium (1% yeast extract, 2% glucose, 2% peptone, 2% agar). In the case of the mutant strain, the medium also contained 0.02% geneticin. Cells were grown in liquid YPD 2%, YPD 0.5% (containing 0.5% glucose instead of 2%) or YPGly (containing 4% glycerol instead of glucose) medium until the exponential phase (OD570 = 0.6), using an orbital shaker at 28 8C and 160 rpm with the ratio of flask volume/medium of 5/1. For cell yield, cells were resuspended at 0.05 mg dry weight/mL and cell mass was determined after 24 h by measuring OD570. 2.2. Chronological lifespan and mutagenic rate Cells were grown to approximately 0.8 mg dry weight/mL. Chronological lifespan was assayed as previously described (Harris et al., 2003). Cells were centrifuged at 4000 rpm for 5 min, resuspended in ultrapure water, and centrifuged again (repeated twice). Washed cells were resuspended in water and incubated at 37 8C for the indicated times. Cell viability was determined by standard dilution plate counts on YPD medium containing 2% (w/v) agar. Cells were also plated on YPGly medium to test strains for the petite formation. Colonies were counted after growth at 28 8C for 3 days. Viability was expressed as the percentage of colonyforming units. The mutation rate caused by ageing was measured as the percentage of colonies on YPGly in relation to YPD plates. 2.3. Intracellular oxidation The oxidant-sensitive probe 20 ,70 -dichlorofluorescein diacetate (DCF) was used to assess intracellular oxidation during ageing. Fluorescence was measured using a PTI (Photo Technology International) spectrofluorimeter set at an excitation wavelength of 504 nm and an emission wavelength of 524 nm (Brennan and Schiestl, 1996; Davidson et al., 1996). Yeast cells (50 mg) were incubated with 10 mM DCF (from a fresh 5 mM stock in ethanol) for 15 min to allow uptake of the
701
probe. Cells were harvested by centrifugation and washed twice with 50 mM phosphate buffer pH 6.0. The pellet was resuspended in 0.5 mL of the same buffer and 1.5 g of glass beads was added. The samples were lysed by three cycles of 1 min agitation on a vortex mixer followed by 1 min on ice. The supernatant, obtained after centrifugation at 25,000 g for 5 min, was diluted sixfold with water and fluorescence was measured. 2.4. Protein carbonylation analysis Yeast extracts were prepared in 50 mM potassium phosphate buffer, pH 7.0, and 0.1 mM EDTA, containing a protease inhibitor cocktail (Complete, Mini, EDTA-free Protease Cocktail Inhibitor Tablets; Boehringer Mannhein). Protein content was estimated according to Lowry et al. (1951), using bovine serum albumin as standard. Protein oxidation was determined by immunodetection of protein carbonyls as previously described (Costa et al., 2002). Proteins were derivatised with 2,4-dinitrophenylhydrazine and slot-blotted (0.1 mg) into Polyvinylidene fluoride membranes (Hybond-PVDF, GE Healthcare, Europe) or separated by 2D-gel electrophoresis (200 mg), using 13 cm immobilized pH 3–10NL gradient (IPG) dry strips (GE Healthcare, Europe) for isoelectric focusing and a 12.5% polyacrylamide gel for SDS-PAGE. After electrophoresis, proteins were stained with Coomassie blue or electroblotted onto a Hybond-ECL membrane (GE Healthcare, Europe). The ECL or PVDF membranes were probed with rabbit IgG anti-DNP (Dako, Glostrup, Denmark) at a 1:5000 dilution as the primary antibody, and goat anti-rabbit IgG–peroxidase (Sigma, St. Louis, MO, USA) at a 1:5000 dilution as the secondary antibody. Immunodetection was performed by chemiluminescence, using a kit from GE Healthcare, Europe (RPN 2109). The membranes were exposed to a Hyperfilm-ECL film (GE Healthcare, Europe) for 15 s to 1 min, and the film was developed. Quantification of carbonyls was performed by densitometry. 2.5. Protein identification Proteins were excised from polyacrylamide gels stained with 0.05% (w/v) Coomassie blue, 0.5% (v/v) acetic acid, 20% (v/v) methanol, and destained with 30% (v/v) methanol. Proteins were digested with trypsin (Promega, Madison). The peptide extracts were concentrated and analyzed using the 4700 Proteomics Analyzer MALDI-TOF/TOF (Applied Biosystems, Foster City, CA, USA) in MS mode. The acquisition, analysis and interpretations were performed as described before (Finehout et al., 2004). 2.6. Enzyme activities and oxygen consumption Extracts for enzymatic determinations were obtained by the disruption of cells with glass beads in 0.1 M Tris–HCl buffer, pH 8.0 (Pereira et al., 2003). Protein was determined as described by Stickland (1951), using bovine serum albumin as standard. Glutathione reductase activity was assayed spectrophotometrically on the basis of NADPH consumption (Gunasekaran et al., 1995; Chouchane and Snow, 2001). One unit of glutathione reductase activity is defined as the consumption of 1 mmol of NADPH for 1 min. Glucose-6-phosphate-dehydrogenase activity was measured according to Glock and McLean (1953). One unit of glucose-6-phosphatedehydrogenase activity is defined as the formation of 1 mmol of NADPH for 1 min. Pyruvate kinase activity was determined using a lactate dehydrogenase coupled reaction assay by measuring the decrease in absorbance at 340 nm resulting from NADH oxidation (Bergmeyer, 1974). One unit of pyruvate kinase activity is defined as the consumption of 1 mmol of NADH for 1 min. Aconitase activity was determined using an isocitrate dehydrogenase NADPH-dependent coupled reaction assay by measuring the increase in absorbance at 340 nm resulting from NADPH formation (Gardner et al., 1995). One unit of aconitase activity is defined as the production of 1 mmol of NADPH for 1 min. Oxygen consumption was followed at 25 8C in cell suspensions at 10 mg dry weight/mL incubated in 108 mM glucose and 50 mM Tris–HCl (pH 4.5) using a computer-interfaced Clark electrode operating in an air-tight chamber with continuous stirring (Bonawitz et al., 2006). 2.7. Statistical analysis Data were expressed as mean values S.D. of at least three independent experiments. Values were compared by Student’s t-test. The latter denotes homogeneity between experimental groups at p < 0.05.
3. Results GSH is the most abundant non-protein thiol that plays a key role on redox homeostasis (Penninckx, 2002). It is synthesized in two ATP-dependent steps catalyzed by gamma-glutamylcysteine synthetase (Gsh1p) and glutathione synthetase (Gsh2p). To evaluate the role of GSH in chronological lifespan extension of calorie-restricted cells, we used the gsh1 mutant strain that is
702
S.C. Mannarino et al. / Mechanisms of Ageing and Development 129 (2008) 700–705
Fig. 1. Glutathione deficiency increases chronological lifespan of calorie-restricted cells. Chronological lifespan of non-CR (A) or CR cells (B). Wild type (white bars) and gsh1 mutant cells (black bars) were grown in YPD 2% (A) or YPD 0.5% medium (B), washed with water and incubated in water at 37 8C up to 24 h. Cellular viability was measured by standard dilution plate counts and expressed as the percentage of the colony-forming units at time 0 h. (C) Glutathione reductase (Glr) and (D) glucose-6-phosphatedehydrogenase (G6PDH) activities were measured in CR cells immediately before and after 6 h of ageing. Values are mean S.D. of three independent experiments. *p < 0.05 (gsh1 vs. WT).
deficient in glutathione biosynthesis. This same strain has been used in other works of our group and when a control experiment is done through the addition of GME (glutathione mono-ethyl ester), the gsh1 mutant recovers the control strain phenotype (Gomes et al., 2002; Adamis et al., 2004, 2007). Thus, we assume that there is not any secondary mutation in our strain that may masquerade the real function of glutathione in the ageing process under calorie restriction. Yeast cells were grown in culture medium containing 2 or 0.5% glucose (to simulate CR), transferred into water and incubated at 37 8C to accelerate ageing process. It had been shown that differences in lifespan measured at 30 8C are reproducible at 37 8C (MacLean et al., 2001; Harris et al., 2005; Piper et al., 2006). Under these conditions, chronological lifespan of CR cells was significantly higher than that observed in 2% glucose grown cells, in which both strains presented a similar lifespan (Fig. 1A). However, glutathione deficiency increased the longevity of CR cells (Fig. 1B): cellular viability after 6 and 24 h of ageing was 69 and 15% for parental cells and 88 and 27% for gsh1 mutants. This was unexpected since the absence of an antioxidant defense usually decreases cellular longevity (Piper, 2006). Next, the activity of some antioxidant enzymes was measured. A deficiency in some antioxidant system, like GSH, might be overcomed by an increase in the remaining antioxidants, like catalase and glucose-6-phosphate dehydrogenase (G6PDH), explaining why gsh1 strain showed a higher lifespan than WT. Catalase is considered the first line of cell defense against oxidative injury. G6PDH is involved in the pentose phosphate pathway, and as such is crucial for the production of cellular reducing power in the form of NADPH. The enzymes glutathione reductase (Glr) and thioredoxin reductase both require NADPH as a reductant to reduce oxidized glutathione (GSSG) and thioredoxin. As shown in Fig. 1C and D, the activity of Glr or G6PDH was not affected by glutathione deficiency, either at t0 or in cells aged for 6 h. Total catalase and superoxide dismutase activities were also similar in both strains (results not shown).
To assess if the increase in chronological lifespan of CR gsh1 mutant cells was correlated with lower levels of oxidative stress markers, we evaluated ROS production, using the fluorescent probe 20 ,70 -dichlorofluorescein diacetate, and protein carbonylation. In both WT and gsh1 mutant cells, intracellular oxidation increased during cell ageing (Fig. 2A). However, the increase in ROS levels was lower in the gsh1 strain. The analysis of protein carbonylation also showed lower levels in aged gsh1 mutant cells, compared to aged WT cells (Fig. 2B). These results show that the increase in chronological lifespan of GSH deficient cells is related to a diminished ROS production and protein oxidation. Recent evidences have shown that a few specific proteins are carbonylated in aged cells, including molecular chaperones (Hsp60 and Hsp70) and enzymes involved in glucose metabolism such as enolase, glyceraldehyde-3-P dehydrogenase, fructose-1,6-biphosphate, aldolase, pyruvate decarboxylase, and alcohol dehydrogenase (Reverter-Branchat et al., 2004). Therefore we decided to search for differences in carbonylation of specific proteins in WT and gsh1 mutant strains, using 2D-gel electrophoresis. The results showed that GSH deficiency decreased the carbonyl content of a few specific proteins in aged cells (Fig. 2C and D; indicated by arrows). These proteins were excised from a 2D silver stained replica gel and identified by mass spectrometry, as pyruvate decarboxylase, pyruvate kinase and alcohol dehydrogenase (Table 1). These proteins are functionally associated with glycolysis/fermentation. Protein carbonylation decreases enzyme activity and higher levels of alcohol dehydrogenase (Adh1) have been correlated with lifespan extension (Reverter-Branchat et al., 2007). Therefore, the decrease in Adh1 carbonylation may contribute to the increase in life expectancy of gsh1 mutant cells, compared to WT (Fig. 1A). In our experiments, when cells were grown under calorierestricted conditions (0.5% glucose), the gsh1 strain displayed about 51% higher cell yield. However, under strictly fermenting (2% glucose) or respiring (4% glycerol) conditions, the gsh1 strain
S.C. Mannarino et al. / Mechanisms of Ageing and Development 129 (2008) 700–705
703
Fig. 2. Oxidative stress markers during chronological ageing. Wild type (white bars) and gsh1 mutant cells (black bars) were grown under CR and incubated in water at 37 8C. (A) Intracellular oxidation. Cells were labeled with the 20 ,70 -dichlorofluorescein diacetate probe and lysed. Data are expressed as the fluorescence ratio between 6 and 24 h aged cells and control. (B) Protein carbonylation. Cells were lysed and proteins were derivatised with DNPH and blotted into a PVDF membrane where protein carbonyls were immunodetected. Data are expressed as the protein carbonyls ratio between 24 h aged cells and control. Values are mean S.D. of three independent experiments. **p < 0.01 (gsh1 vs. WT). (C and D) 2D-analysis of protein carbonyls. WT (C) and gsh1 (D) cells were lysed after 24 h ageing, proteins were derivatised with DNPH, separated by two-dimensional gel electrophoresis, Western blotted into a nitrocellulose membrane and carbonyls immunodetected. Both blots were processed at the same time, to allow direct comparisons. The experiment was reproduced three times, using independent samples. A representative blot is shown. The molecular mass is shown on the left.
Table 1 Identification of proteins differentially carbonylated in gsh1 mutants by MALDI-MS. Protein score CI was 100% for all spots. Gel spot
Accession no.
Systematic name
Protien name
Protien function
MW (Da)
pl
No. peptides matched
1 2 3
gij6323073 gij51013665 gij6324486
YLR044C YAL038W YOL086C
PdC1p Cdc19p Adh1p
Pyruvate decarboxylase Pyruvate kinase Alcohol dehydrogenase
61456.6 54909.5 36825.7
5.8 7.6 6.2
25 16 17
showed a slightly lower cell yield. Aiming to investigate if glutathione deficiency affected energy metabolism, we measured pyruvate kinase and aconitase activity and oxygen consumption in cells grown under calorie-restricted conditions (Fig. 3A). The results showed an increased activity of the glycolytic enzyme pyruvate kinase in calorie-restricted gsh1 mutant cells, suggesting that GSH deficiency favors a faster ATP production by fermentative growth. This would explain the increase in the cell yield of gsh1 mutant cells grown in 0.5% glucose medium. In agreement, aconitase activity and oxygen consumption were 40 and 21%
lower, respectively, in the gsh1 mutant (Fig. 3A and B). This decrease in mitochondrial function is consistent with the lower ROS production observed in aged gsh1 mutant cells, leading to an increased lifespan (Figs. 1A and 2A). Despite the involvement of GSH metabolism in the formation and maintenance of iron–sulfur clusters in mitochondrial enzymes (Sonq et al., 2006), the gsh1 null mutant strain showed a higher aconitase activity than WT when cells were grown under restricted respiratory conditions (data not shown). Therefore, the decrease in aconitase activity under CR reflects changes in metabolic fluxes in detriment of respiration.
Fig. 3. Glutathione deficiency reduces respiration rate of calorie-restricted cells. (A and B) Wild type (white bars) and gsh1 mutant cells (black bars) were grown under CR. Pyruvate kinase and aconitase activities (A) were measured in cells taken at t0 (before incubation at 37 8C), using protein extracts. Oxygen consumption (B) was measured in cell suspensions using a Clark electrode. Values are mean S.D. of three independent experiments. *p < 0.05.
704
S.C. Mannarino et al. / Mechanisms of Ageing and Development 129 (2008) 700–705
Fig. 4. Glutathione deficiency increases mutagenesis during chronological ageing. Wild type (white bars) and gsh1 mutant cells (black bars) were grown under CR, washed with water and incubated in water at 37 8C. (A) Mutagenic rate was measured as described in Section 2. (B) Competition assay. The same number of cells was taken from 72 haged cultures, mixed and incubated in fresh YPD 2% media. Cellular viability was followed at 0, 24 and 48 h. The percentage of viable cells of each strain was obtained by colony counting on YPD 2% and YPD 2% + geneticine plates. Values are mean S.D. of three independent experiments. **p < 0.01.
It was previously shown that respiration adapted yeast cells display an increased chronological lifespan, compared to cells pregrown on fermenting medium (MacLean et al., 2001; Harris et al., 2005; Piper et al., 2006). However, according to our results, CR gsh1 cells presented a decreased mitochondrial respiration but a longer chronological lifespan. To evaluate if this advantage for survival might only be a short-term benefit, we measured mitochondrial mutagenesis as the rate of petite formation during ageing. In cells aged for 6 h, the percentage of petites was similar to the observed at t0 (data not shown). However, as shown in Fig. 4A, GSH deficiency increased the mutation rate after 24 h of cell ageing, indicating that, although calorie-restricted gsh1 strain lives longer than WT, it accumulates mutations that are prejudicial for its population. We also investigated how aged cells adapted and recovered when inoculated in a fresh media. For both strains, cellular viability after 3 days of ageing are almost null, but when aged cells were reinoculated in fresh YPD 2% media, surviving cells were able to recover and grow. To analyze how these aged cultures compete for nutrients in the same environment, we mixed the same amount of WT and gsh1 mutant cells in fresh medium and monitored colonyforming units during recovery. To discriminate between the two strains, the mixed culture was plated on YPD 2% supplemented or not with geneticine (only gsh1 mutant cells are able to grow on both). As seen in Fig. 4B, WT strain dominated the environment, overwhelming the gsh1 strain. These results corroborate that GSH deficient cells have a short-term advantage but aged cells accumulate higher levels of mitochondrial DNA damage and are unable to compete against WT cells. 4. Discussion In this study, we showed that GSH deficiency decreased the metabolic switch from fermentation to respiration in CR cells. Nevertheless, chronological lifespan increased in CR gsh1 mutant cells. Under these conditions, the intracellular oxidation and protein carbonylation associated with the ageing process was decreased in gsh1 mutant cells. This was unexpected, since GSH is an important antioxidant molecule and lifespan is usually shortened in antioxidant deficient cells (Piper, 2006). The 2Danalysis showed that the carbonylation of pyruvate kinase, pyruvate decarboxylase and alcohol dehydrogenase 1 (Adh1), three proteins previously shown to be oxidized in aged cells (Reverter-Branchat et al., 2004), decreased in aged gsh1 mutant cells. Adh1 catalyses the conversion of acetaldehyde to ethanol, regenerating NAD+. It was previously shown that NAD+/NADH balancing mediated by this enzyme is important to chronological lifespan (Reverter-Branchat et al., 2007). The protection of Adh1 during ageing of gsh1 mutant cells may, therefore, contribute to lifespan extension.
The decrease in ROS production during ageing of gsh1 mutant cells was correlated with a decrease in aerobic metabolism, as indicated by a reduction in oxygen consumption and aconitase activity. In contrast, the activity of pyruvate kinase, one of the most controlled glycolytic enzyme, increased. Despite this short-term advantage, GSH deficiency increased the mitochondrial genome mutation rate during ageing, compromising the mutant at long term. This is consistent with the instability of the mitochondrial genome exhibited by gsh1 mutant cells (Lee et al., 2001). In agreement, aged WT cells outlasted aged gsh1 mutant cells in direct competition assays in a fresh medium. Since GSH deficiency decreased intracellular oxidation in aged cells, it seems unlikely that ROS mediate mitochondrial DNA damage. The carbonylation of pyruvate kinase, pyruvate decarboxylase and alcohol dehydrogenase in aged WT cells suggests that this strain may control its metabolism through inactivation of these proteins. Diverse published data shows that protein carbonylation is an irreversible modification that decreases protein activity. Therefore, protein carbonylation in aged WT cells impairs enzyme activity. As a consequence, the glycolytic flux would decrease and the desired respiration rate would be maintained to achieve a higher lifespan (compared to glucose repressed cells) and all the benefits that CR can provide. Although it had been reported that respiration is not required for the longevity benefits of CR in yeast (Kaeberlein et al., 2005), our results suggest that respiration confers long-term benefits of lifespan extension. Herker et al. (2004) showed that apoptosis in yeast confers a selective advantage for this organism, by eliminating defective cells from culture and saving nutrients for the remaining cells. When yca1 null mutants, deficient in the yeast metacaspase, have to compete against WT cells, the latter show better survivals in the end. Recently it was shown by Gomes et al. (2008) that a glutaredoxin system, which is dependent on GSH, is involved in apoptosis signaling under cadmium stress. Whether apoptosis signaling is compromised in the gsh1 mutant strain, affecting its ability to remove undesired or damaged cells during the ageing process, is an important issue to be investigated in further studies. In summary, our results show that glutathione deficiency increases chronological lifespan of CR cells. This lifespan extension is correlated with lower levels of oxidative stress markers, including a decrease in ROS and Adh1 carbonylation that was associated with a reduction in respiration metabolism. However, gsh1 null mutants accumulate high levels of mitochondrial DNA damage during cell ageing and are unable to compete with aged WT cells. In the WT strain, despite the higher respiration rate and the more oxidized intracellular environment in aged cells, cells are able to prevent mutations and are fit to compete with gsh1 mutant strain, which guarantee a long-term advantage.
S.C. Mannarino et al. / Mechanisms of Ageing and Development 129 (2008) 700–705
Acknowledgements We thank Prof. Reginaldo Menezes for expert assistance in oxygen consumption analyses. This project was supported by CAPES (GRICES/PROCAD), CNPq, FAPERJ and FAPESP (grant 0410067/6). References Adamis, P.D., Gomes, D.S., Pinto, M.L., Panek, A.D., Eleutherio, E.C., 2004. The role of glutathione transferases in cadmium stress. Toxicol. Lett. 154, 81–88. Adamis, P.D., Panek, A.D., Eleutherio, E.C., 2007. Vacuolar compartmentation of the cadmium–glutathione complex protects Saccharomyces cerevisiae from mutagenesis. Toxicol. Lett. 173, 1–7. Balaban, R.S., Nemoto, S., Finkel, T., 2005. Mitochondria, oxidants and aging. Cell 120, 483–495. Barja, G., 2002. Endogenous oxidative stress: relationship to aging, longevity and caloric restriction. Ageing Res. Rev. 1, 397–411. Barros, M.H., Bandy, B., Tahara, E.B., Kowaltowski, A.J., 2004. Higher respiratory activity decreases mitochondrial reactive oxygen release and increases life span in Saccharomyces cerevisiae. J. Biol. Chem. 279, 49883–49888. Bergmeyer, H.U., 1974. Methods of Enzymatic Analysis, 2nd ed., vol. I. Academic Press, pp. 465. Bitterman, K.J., Medvedik, O., Sinclair, D.A., 2003. Longevity regulation in Saccharomyces cerevisiae: linking metabolism, genome stability, and heterochromatin. Microbiol. Mol. Biol. Rev. 67, 376–399. Bonawitz, N.D., Rodeheffer, M.S., Shadel, G.S., 2006. Defective mitochondrial gene expression results in reactive oxygen species-mediated inhibition of respiration and reduction of yeast life span. Mol. Cell. Biol. 26, 4818–4829. Brennan, R.J., Schiestl, R.H., 1996. Cadmium is an inducer of oxidative stress in yeast. Mutat. Res. 356, 171–178. Chouchane, S., Snow, E., 2001. In vitro effect of arsenical compounds on glutathionerelated enzymes. Chem. Res. Toxicol. 14, 517–522. Costa, V., Amorim, M.A., Quintanilha, A., Moradas-Ferreira, P., 2002. Hydrogen peroxide-induced carbonylation of key metabolic enzymes in Saccharomyces cerevisiae: the involvement of the oxidative stress response regulators Yap1 and Skn7. Free Radic. Biol. Med. 33, 1507–1515. Davidson, J.F., Whyte, B., Bissinger, P.H., Schiestl, R.H., 1996. Oxidative stress is involved in heat-induced death in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. U.S.A. 93, 5116–5121. Dillin, A., Hsu, A.L., Arantes-Oliveira, N., Lehrer-Graiwer, J., Hsin, H., Fraser, A.G., Kamath, R.S., Ahringer, J., Kenyon, C., 2002. Rates of behavior and aging specified by mitochondrial function during development. Science 298, 2398–2401. Fabrizio, P., Gattazzo, C., Battistella, L., Wei, M., Cheng, C., 2005. Sir2 blocks extreme life-span extension. Cell 123, 655–667. Fabrizio, P., Liou, L.L., Moy, V.N., Diaspro, A., Valentine, J., 2003. SOD2 functions downstream of Sch9 to extend longevity in yeast. Genetics 163, 35–46. Fabrizio, P., Longo, V.D., 2003. The chronological life span of Saccharomyces cerevisiae. Aging Cell 2, 73–81. Feng, J., Bussiere, F., Hekimi, S., 2001. Mitochondrial electron transport is a key determinant of life span in Caenorhabditis elegans. Dev. Cell 1, 633–644. Finehout, E.J., Franck, Z., Lee, K.H., 2004. Towards two-dimensional electrophoresis mapping of the cerebrospinal fluid proteome from a single individual. Electrophoresis 25, 2564–2575. Gardner, P.R., Raineri, I., Epstein, L.B., White, C.W., 1995. Superoxide radical and iron modulate aconitase activity in mammalian cells. J. Biol. Chem. 270, 13399– 13405. Glock, G.E., McLean, P., 1953. Further studies on the properties and assay of glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase of rat liver. Biochem. J. 55, 400–408. Gomes, D.S., Fragoso, L.C., Riger, C.J., Panek, A.D., Eleutherio, E.C., 2002. Regulation of cadmium uptake by Saccharomyces cerevisiae. Biochim. Biophys. Acta 1573, 21– 25. Gomes, D.S., Pereira, M.D., Panek, A.D., Andrade, L.R., Eleutherio, E.C., 2008. Apoptosis as a mechanism for removal of mutated cells of Saccharomyces cerevisiae: the role of Grx2 under cadmium exposure. Biochim. Biophys. Acta 1780, 160–166. Gunasekaran, S., Imbayagwo, M., McDonald, L., 1995. Influence of carbon and nitrogen sources on glutathione catabolic enzymes in Candida albicans during dimorphism. Mycopathologia 131, 93–97. Harris, N., Costa, V., MacLean, M., Mollapour, M., Moradas-Ferreira, P., Piper, P.W., 2003. Mn-Sod overexpression extends the yeast chronological (G0) life span, but acts independently of Sir2p histone deacetylase to shorten the replicative life span of dividing cells. Free Radic. Biol. Med. 34, 1599–1606.
705
Harris, N., Bachler, M., Costa, V., Mollapour, M., Moradas-Ferreira, P., Piper, P.W., 2005. Overexpressed Sod1p acts either to reduce or to increase the lifespans and stress resistance of yeast, depending on whether it is Cu-deficient or an active Cu, Zn-superoxide dismutase. Aging Cell 4, 41–52. Herker, E., Jungwirth, H., Lehmann, K.A., Maldener, C., Fro¨hlich, K.U., Wissing, S., Bu¨ttner, S., Fehr, M., Sigrist, S., Madeo, F., 2004. Chronological aging leads to apoptosis in yeast. J. Cell Biol. 164, 501–507. Jazwinski, S.M., 1999. Longevity, genes, and aging: a view provided by a genetic model system. Exp. Gerontol. 34, 1–6. Jiang, J.C., Jaruga, E., Repnevskaya, M.V., Jazwinski, S.M., 2000. An intervention resembling caloric restriction prolongs life span and retards aging in yeast. FASEB J. 14, 2135–2137. Kaeberlein, M., Burtner, C.R., Kennedy, B.K., 2007. Recent developments in yeast aging. PLoS Genet. 3, e84. Kaeberlein, M., Hu, D., Kerr, E.O., Tsuchiya, M., Westman, E.A., 2005. Increased life span due to calorie restriction in respiratory-deficient yeast. PLoS Genet. 1, e69. Lee, J.C., Straffon, M.J., Jang, T.Y., Higgins, V.J., Grant, C.M., Dawes, I.W., 2001. The essential and ancillary role of glutathione in Saccharomyces cerevisiae analysed using a grande gsh1 disruptant strain. FEMS Yeast Res. 1, 57–65. Lin, S.J., Defossez, P.A., Guarente, L., 2000. Requirement of NAD and SIR2 for life-span extension by calorie restriction in Saccharomyces cerevisiae. Science 289, 2126– 2128. Lin, S.J., Kaeberlein, M., Andalis, A.A., Sturtz, L.A., Defossez, P.A., Culotta, V.C., Fink, G.R., Guarente, L., 2002. Calorie retriction extends Saccharomyces cerevisiae lifespan by increasing respiration. Nature 418, 344–348. Lin, S.J., Ford, E., Haigis, M., Liszt, G., Guarente, L., 2004. Calorie restriction extends yeast life span by lowering the level of NADH. Genes Dev. 18, 12–16. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, L.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275. MacLean, M.J., Harris, N., Piper, P.W., 2001. Chronological life span of stationary phase yeast cells: a model for investigating the factors that might influence the ageing of postmitotic tissues in higher organisms. Yeast 18, 499–509. Merry, B.J., 2004. Oxidative stress and mitochondrial function with aging—the effects of calorie restriction. Aging Cell 3, 7–12. Penninckx, M.J., 2002. An overview of glutathione in Saccharomyces versus nonconventional yeasts. FEMS Yeast Res. 2, 295–305. Pereira, M.D., Herdeiro, R.S., Fernandes, P.N., Eleutherio, E.C.A., Panek, A.D., 2003. Targets of oxidative stress in yeast sod mutants. Biochim. Biophys. Acta 1620, 245–251. Piper, P.W., 2006. Long-lived yeast as a model for ageing research. Yeast 23, 215– 226. Piper, P.W., Harris, N.L., MacLean, M., 2006. Preadaptation to efficient respiratory maintenance is essential both for maximal longevity and the retention of replicative potential in chronologically aging yeast. Mech. Ageing Dev. 127, 733–740. Powers III, R.W., Kaeberlein, M., Caldwell, S.D., Kennedy, B.K., Fields, S., 2006. Extension of chronological life span in yeast by decreased TOR pathway signaling. Genes Dev. 20, 174–184. Reverter-Branchat, G., Cabiscol, E., Tamarit, J., Ros, J., 2004. Oxidative damage to specific proteins in replicative and chronological-aged Saccharomyces cerevisiae: common targets and prevention by calorie restriction. J. Biol. Chem. 279, 31983–31989. Reverter-Branchat, G., Cabiscol, E., Tamarit, J., Sorolla, M.A., Torre, M.A., Ros, J., 2007. Chronological and replicative life-span extension in Saccharomyces cerevisiae by increased dosage of alcohol dehydrogenase 1. Microbiology 153, 3667–3676. Speakman, J.R., Talbot, D.A., Selman, C., Snart, S., McLaren, J.S., Redman, P., Krol, E., Jackson, D.M., Johnson, M.S., Brand, M.D., 2004. Uncoupled and surviving: individual mice with high metabolism have greater mitochondrial uncoupling and live longer. Aging Cell 3, 87–95. Sohal, R.S., Weindruch, R., 1996. Oxidative stress, caloric restriction, and aging. Science 273, 59–63. Sohal, R.S., 2002. Oxidative stress hypothesis of aging. Free Radic. Biol. Med. 33, 573–574. Sonq, J.Y., Cha, J., Lee, J., Roe, J.H., 2006. Glutathione reductase and a mitochondrial thioredoxin play overlapping roles in maintaining iron–sulfur enzymes in fission yeast. Eukaryot. Cell 5, 1857–1865. Stickland, L.H., 1951. The determination of small quantities of bacteria by means of the Biuret reaction. J. Gen. Microbiol. 5, 698–703. Sto¨ckl, P., Zankl, C., Hu¨tter, E., Unterluggauer, H., Laun, P., Heeren, G., Bogengruber, E., Herndler-Brandstetter, D., Breitenbach, M., Jansen-Du¨rr, P., 2007. Partial uncoupling of oxidative phosphorylation induces premature senescence in human fibroblasts and yeast mother cells. Free Radic. Biol. Med. 43, 947–958. Thomas, J.A., Mallis, R.J., 2001. Aging and oxidation of reactive protein sulfhydryls. Exp. Gerontol. 36, 1519–1526.
Contents lists available at ScienceDirect
Mechanisms of Ageing and Development journal homepage: www.elsevier.com/locate/mechagedev
Glutathione is necessary to ensure benefits of calorie restriction during ageing in Saccharomyces cerevisiae Se´rgio C. Mannarino a,*, Maria A. Amorim b,c, Marcos D. Pereira a, Pedro Moradas-Ferreira b,c, Anita D. Panek a, Vı´tor Costa b,c, Elis C.A. Eleutherio a a b c
Departamento de Bioquı´mica, Instituto de Quı´mica, UFRJ, 21941-909 Rio de Janeiro, RJ, Brazil IBMC, Rua do Campo Alegre 823, 4150-180 Porto, Portugal ICBAS, Departamento de Biologia Molecular, Universidade do Porto, Porto, Portugal
A R T I C L E I N F O
A B S T R A C T
Article history: Received 28 April 2008 Received in revised form 11 August 2008 Accepted 8 September 2008 Available online 18 September 2008
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. ß 2008 Elsevier Ireland Ltd. All rights reserved.
Keywords: Saccharomyces cerevisiae Chronological ageing Glutathione Calorie restriction
1. Introduction Low-calorie diets known as calorie restriction (CR) extend the lifespan of organisms ranging from yeast to mammals. The molecular mechanisms by which CR slows ageing have been associated with the activation of Sir2-family proteins, modulation of nutrient signaling pathways, decreased production of reactive oxygen species (ROS) and accumulation of oxidative damages (Sohal and Weindruch, 1996; Barja, 2002; Sohal, 2002; Merry, 2004). The budding yeast Saccharomyces cerevisiae has been widely used as a model system to study the mechanisms of modulation of two different types of lifespan: replicative lifespan, measured as the number of daughter cells produced by a mother cell before senescence and chronological lifespan, the length of time a yeast cell can survive in a nondividing state (Jazwinski, 1999; Fabrizio and Longo, 2003; Bitterman et al., 2003; Piper, 2006). In both ageing models, nutrient signaling has been linked to longevity (Kaeberlein et al., 2007). In the lab, yeast cells are typically grown in media containing high levels of glucose (2%) and amino acids. However, independent studies have shown that
* Corresponding author. Tel.: +55 21 2562 7735; fax: +55 21 2562 7735. E-mail address: [email protected] (S.C. Mannarino). 0047-6374/$ – see front matter ß 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mad.2008.09.001
replicative and chronological lifespan can be increased by reducing either glucose or amino acid concentration (or both) (ReverterBranchat et al., 2004; Fabrizio et al., 2003, 2005; Jiang et al., 2000; Lin et al., 2000; Powers et al., 2006). A reduction in glucose concentration results in a metabolic shift from fermentation to respiration (Lin et al., 2002). While it is often incorrectly assumed that respiration and ROS production are directly proportional, the biological consequences of changes in respiration on ROS production and longevity are complex and far from being completely understood (Balaban et al., 2005; Speakman et al., 2004). Lifespan extension has been associated to both increase (Barros et al., 2004; Speakman et al., 2004; Bonawitz et al., 2006) and decrease (Feng et al., 2001; Dillin et al., 2002) in respiration rate. In yeast, Barros et al. (2004) suggested that CR increases mitochondrial respiration but decreases the ratio (ROS released)/ (O2 consumed) by reducing the leakage of electrons from the respiratory chain. A more recent study shows, however, that the partial uncoupling of oxidative phosphorylation in human fibroblasts and yeast mother cells increases ROS production and induces premature senescence (Sto¨ckl et al., 2007). The central regulatory role of mitochondrial metabolism in both replicative and chronological ageing in yeast is further supported by different studies. Yeast cells pre-grown on a respiratory carbon source, compared to glucose grown cells, have a higher chron-
S.C. Mannarino et al. / Mechanisms of Ageing and Development 129 (2008) 700–705
ological longevity. This prior adaptation to efficient respiratory maintenance also ensures that such chronological aged cells still display a full replicative potential should they reenter the cell cycle (Piper et al., 2006). Indeed, yeast longevity is extended by overexpression of the HAP4 transcription factor, which increases the expression of numerous respiratory genes (Lin et al., 2002; Piper et al., 2006), or overexpression of mitochondrial NADdependent dehydrogenases (Lin et al., 2004). Mitochondrial dysfunction associated with ageing increases ROS production leading to the accumulation of oxidative damages to proteins, lipids and DNA. In agreement, studies in yeast and other model organisms showed that lifespan is shortened in antioxidant deficient cells and the overexpression of antioxidant defenses delays ageing (reviewed by Piper, 2006). Some studies have suggested that CR functions to extend lifespan by increasing resistance to ROS (Sohal and Weindruch, 1996). Glutathione (gamma-glutamylcysteinylglycine; GSH) is a low-molecular-mass thiol with proposed functions in many cellular processes including protection against xenobiotics, carcinogens and ROS, being used as a co-factor for glutathione peroxidase or protecting cysteine residues in proteins through S-thiolation (Thomas and Mallis, 2001). The biological importance of GSH is dependent upon the redox-active free sulfydryl moiety of its cysteine residue. In this work, we investigated the importance of the antioxidant metabolite glutathione to ensure full benefits of CR in cellular longevity. We observed that yeast cells deficient in glutathione production (gsh1 strain) have an increased chronological lifespan, compared to wild-type cells, and present lower levels of oxidative stress markers. We noted a difference between both strains regarding their energy metabolism, where respiration rate is higher in the gsh1 mutant strain. However, aged gsh1 mutant cells show a higher mitochondrial mutagenic rate and are not able to compete with aged wild type when they are in the same environment. 2. Materials and methods 2.1. Yeast strains and growth conditions Saccharomyces cerevisiae wild-type strain BY4741 (MATa; his3; leu2; met15; ura3) and its isogenic mutant gsh1 harboring the gene GSH1 interrupted by the gene KanMX4, were acquired from Euroscarf, Frankfurt, Germany. Stocks of yeast strains were maintained on solid YPD 2% medium (1% yeast extract, 2% glucose, 2% peptone, 2% agar). In the case of the mutant strain, the medium also contained 0.02% geneticin. Cells were grown in liquid YPD 2%, YPD 0.5% (containing 0.5% glucose instead of 2%) or YPGly (containing 4% glycerol instead of glucose) medium until the exponential phase (OD570 = 0.6), using an orbital shaker at 28 8C and 160 rpm with the ratio of flask volume/medium of 5/1. For cell yield, cells were resuspended at 0.05 mg dry weight/mL and cell mass was determined after 24 h by measuring OD570. 2.2. Chronological lifespan and mutagenic rate Cells were grown to approximately 0.8 mg dry weight/mL. Chronological lifespan was assayed as previously described (Harris et al., 2003). Cells were centrifuged at 4000 rpm for 5 min, resuspended in ultrapure water, and centrifuged again (repeated twice). Washed cells were resuspended in water and incubated at 37 8C for the indicated times. Cell viability was determined by standard dilution plate counts on YPD medium containing 2% (w/v) agar. Cells were also plated on YPGly medium to test strains for the petite formation. Colonies were counted after growth at 28 8C for 3 days. Viability was expressed as the percentage of colonyforming units. The mutation rate caused by ageing was measured as the percentage of colonies on YPGly in relation to YPD plates. 2.3. Intracellular oxidation The oxidant-sensitive probe 20 ,70 -dichlorofluorescein diacetate (DCF) was used to assess intracellular oxidation during ageing. Fluorescence was measured using a PTI (Photo Technology International) spectrofluorimeter set at an excitation wavelength of 504 nm and an emission wavelength of 524 nm (Brennan and Schiestl, 1996; Davidson et al., 1996). Yeast cells (50 mg) were incubated with 10 mM DCF (from a fresh 5 mM stock in ethanol) for 15 min to allow uptake of the
701
probe. Cells were harvested by centrifugation and washed twice with 50 mM phosphate buffer pH 6.0. The pellet was resuspended in 0.5 mL of the same buffer and 1.5 g of glass beads was added. The samples were lysed by three cycles of 1 min agitation on a vortex mixer followed by 1 min on ice. The supernatant, obtained after centrifugation at 25,000 g for 5 min, was diluted sixfold with water and fluorescence was measured. 2.4. Protein carbonylation analysis Yeast extracts were prepared in 50 mM potassium phosphate buffer, pH 7.0, and 0.1 mM EDTA, containing a protease inhibitor cocktail (Complete, Mini, EDTA-free Protease Cocktail Inhibitor Tablets; Boehringer Mannhein). Protein content was estimated according to Lowry et al. (1951), using bovine serum albumin as standard. Protein oxidation was determined by immunodetection of protein carbonyls as previously described (Costa et al., 2002). Proteins were derivatised with 2,4-dinitrophenylhydrazine and slot-blotted (0.1 mg) into Polyvinylidene fluoride membranes (Hybond-PVDF, GE Healthcare, Europe) or separated by 2D-gel electrophoresis (200 mg), using 13 cm immobilized pH 3–10NL gradient (IPG) dry strips (GE Healthcare, Europe) for isoelectric focusing and a 12.5% polyacrylamide gel for SDS-PAGE. After electrophoresis, proteins were stained with Coomassie blue or electroblotted onto a Hybond-ECL membrane (GE Healthcare, Europe). The ECL or PVDF membranes were probed with rabbit IgG anti-DNP (Dako, Glostrup, Denmark) at a 1:5000 dilution as the primary antibody, and goat anti-rabbit IgG–peroxidase (Sigma, St. Louis, MO, USA) at a 1:5000 dilution as the secondary antibody. Immunodetection was performed by chemiluminescence, using a kit from GE Healthcare, Europe (RPN 2109). The membranes were exposed to a Hyperfilm-ECL film (GE Healthcare, Europe) for 15 s to 1 min, and the film was developed. Quantification of carbonyls was performed by densitometry. 2.5. Protein identification Proteins were excised from polyacrylamide gels stained with 0.05% (w/v) Coomassie blue, 0.5% (v/v) acetic acid, 20% (v/v) methanol, and destained with 30% (v/v) methanol. Proteins were digested with trypsin (Promega, Madison). The peptide extracts were concentrated and analyzed using the 4700 Proteomics Analyzer MALDI-TOF/TOF (Applied Biosystems, Foster City, CA, USA) in MS mode. The acquisition, analysis and interpretations were performed as described before (Finehout et al., 2004). 2.6. Enzyme activities and oxygen consumption Extracts for enzymatic determinations were obtained by the disruption of cells with glass beads in 0.1 M Tris–HCl buffer, pH 8.0 (Pereira et al., 2003). Protein was determined as described by Stickland (1951), using bovine serum albumin as standard. Glutathione reductase activity was assayed spectrophotometrically on the basis of NADPH consumption (Gunasekaran et al., 1995; Chouchane and Snow, 2001). One unit of glutathione reductase activity is defined as the consumption of 1 mmol of NADPH for 1 min. Glucose-6-phosphate-dehydrogenase activity was measured according to Glock and McLean (1953). One unit of glucose-6-phosphatedehydrogenase activity is defined as the formation of 1 mmol of NADPH for 1 min. Pyruvate kinase activity was determined using a lactate dehydrogenase coupled reaction assay by measuring the decrease in absorbance at 340 nm resulting from NADH oxidation (Bergmeyer, 1974). One unit of pyruvate kinase activity is defined as the consumption of 1 mmol of NADH for 1 min. Aconitase activity was determined using an isocitrate dehydrogenase NADPH-dependent coupled reaction assay by measuring the increase in absorbance at 340 nm resulting from NADPH formation (Gardner et al., 1995). One unit of aconitase activity is defined as the production of 1 mmol of NADPH for 1 min. Oxygen consumption was followed at 25 8C in cell suspensions at 10 mg dry weight/mL incubated in 108 mM glucose and 50 mM Tris–HCl (pH 4.5) using a computer-interfaced Clark electrode operating in an air-tight chamber with continuous stirring (Bonawitz et al., 2006). 2.7. Statistical analysis Data were expressed as mean values S.D. of at least three independent experiments. Values were compared by Student’s t-test. The latter denotes homogeneity between experimental groups at p < 0.05.
3. Results GSH is the most abundant non-protein thiol that plays a key role on redox homeostasis (Penninckx, 2002). It is synthesized in two ATP-dependent steps catalyzed by gamma-glutamylcysteine synthetase (Gsh1p) and glutathione synthetase (Gsh2p). To evaluate the role of GSH in chronological lifespan extension of calorie-restricted cells, we used the gsh1 mutant strain that is
702
S.C. Mannarino et al. / Mechanisms of Ageing and Development 129 (2008) 700–705
Fig. 1. Glutathione deficiency increases chronological lifespan of calorie-restricted cells. Chronological lifespan of non-CR (A) or CR cells (B). Wild type (white bars) and gsh1 mutant cells (black bars) were grown in YPD 2% (A) or YPD 0.5% medium (B), washed with water and incubated in water at 37 8C up to 24 h. Cellular viability was measured by standard dilution plate counts and expressed as the percentage of the colony-forming units at time 0 h. (C) Glutathione reductase (Glr) and (D) glucose-6-phosphatedehydrogenase (G6PDH) activities were measured in CR cells immediately before and after 6 h of ageing. Values are mean S.D. of three independent experiments. *p < 0.05 (gsh1 vs. WT).
deficient in glutathione biosynthesis. This same strain has been used in other works of our group and when a control experiment is done through the addition of GME (glutathione mono-ethyl ester), the gsh1 mutant recovers the control strain phenotype (Gomes et al., 2002; Adamis et al., 2004, 2007). Thus, we assume that there is not any secondary mutation in our strain that may masquerade the real function of glutathione in the ageing process under calorie restriction. Yeast cells were grown in culture medium containing 2 or 0.5% glucose (to simulate CR), transferred into water and incubated at 37 8C to accelerate ageing process. It had been shown that differences in lifespan measured at 30 8C are reproducible at 37 8C (MacLean et al., 2001; Harris et al., 2005; Piper et al., 2006). Under these conditions, chronological lifespan of CR cells was significantly higher than that observed in 2% glucose grown cells, in which both strains presented a similar lifespan (Fig. 1A). However, glutathione deficiency increased the longevity of CR cells (Fig. 1B): cellular viability after 6 and 24 h of ageing was 69 and 15% for parental cells and 88 and 27% for gsh1 mutants. This was unexpected since the absence of an antioxidant defense usually decreases cellular longevity (Piper, 2006). Next, the activity of some antioxidant enzymes was measured. A deficiency in some antioxidant system, like GSH, might be overcomed by an increase in the remaining antioxidants, like catalase and glucose-6-phosphate dehydrogenase (G6PDH), explaining why gsh1 strain showed a higher lifespan than WT. Catalase is considered the first line of cell defense against oxidative injury. G6PDH is involved in the pentose phosphate pathway, and as such is crucial for the production of cellular reducing power in the form of NADPH. The enzymes glutathione reductase (Glr) and thioredoxin reductase both require NADPH as a reductant to reduce oxidized glutathione (GSSG) and thioredoxin. As shown in Fig. 1C and D, the activity of Glr or G6PDH was not affected by glutathione deficiency, either at t0 or in cells aged for 6 h. Total catalase and superoxide dismutase activities were also similar in both strains (results not shown).
To assess if the increase in chronological lifespan of CR gsh1 mutant cells was correlated with lower levels of oxidative stress markers, we evaluated ROS production, using the fluorescent probe 20 ,70 -dichlorofluorescein diacetate, and protein carbonylation. In both WT and gsh1 mutant cells, intracellular oxidation increased during cell ageing (Fig. 2A). However, the increase in ROS levels was lower in the gsh1 strain. The analysis of protein carbonylation also showed lower levels in aged gsh1 mutant cells, compared to aged WT cells (Fig. 2B). These results show that the increase in chronological lifespan of GSH deficient cells is related to a diminished ROS production and protein oxidation. Recent evidences have shown that a few specific proteins are carbonylated in aged cells, including molecular chaperones (Hsp60 and Hsp70) and enzymes involved in glucose metabolism such as enolase, glyceraldehyde-3-P dehydrogenase, fructose-1,6-biphosphate, aldolase, pyruvate decarboxylase, and alcohol dehydrogenase (Reverter-Branchat et al., 2004). Therefore we decided to search for differences in carbonylation of specific proteins in WT and gsh1 mutant strains, using 2D-gel electrophoresis. The results showed that GSH deficiency decreased the carbonyl content of a few specific proteins in aged cells (Fig. 2C and D; indicated by arrows). These proteins were excised from a 2D silver stained replica gel and identified by mass spectrometry, as pyruvate decarboxylase, pyruvate kinase and alcohol dehydrogenase (Table 1). These proteins are functionally associated with glycolysis/fermentation. Protein carbonylation decreases enzyme activity and higher levels of alcohol dehydrogenase (Adh1) have been correlated with lifespan extension (Reverter-Branchat et al., 2007). Therefore, the decrease in Adh1 carbonylation may contribute to the increase in life expectancy of gsh1 mutant cells, compared to WT (Fig. 1A). In our experiments, when cells were grown under calorierestricted conditions (0.5% glucose), the gsh1 strain displayed about 51% higher cell yield. However, under strictly fermenting (2% glucose) or respiring (4% glycerol) conditions, the gsh1 strain
S.C. Mannarino et al. / Mechanisms of Ageing and Development 129 (2008) 700–705
703
Fig. 2. Oxidative stress markers during chronological ageing. Wild type (white bars) and gsh1 mutant cells (black bars) were grown under CR and incubated in water at 37 8C. (A) Intracellular oxidation. Cells were labeled with the 20 ,70 -dichlorofluorescein diacetate probe and lysed. Data are expressed as the fluorescence ratio between 6 and 24 h aged cells and control. (B) Protein carbonylation. Cells were lysed and proteins were derivatised with DNPH and blotted into a PVDF membrane where protein carbonyls were immunodetected. Data are expressed as the protein carbonyls ratio between 24 h aged cells and control. Values are mean S.D. of three independent experiments. **p < 0.01 (gsh1 vs. WT). (C and D) 2D-analysis of protein carbonyls. WT (C) and gsh1 (D) cells were lysed after 24 h ageing, proteins were derivatised with DNPH, separated by two-dimensional gel electrophoresis, Western blotted into a nitrocellulose membrane and carbonyls immunodetected. Both blots were processed at the same time, to allow direct comparisons. The experiment was reproduced three times, using independent samples. A representative blot is shown. The molecular mass is shown on the left.
Table 1 Identification of proteins differentially carbonylated in gsh1 mutants by MALDI-MS. Protein score CI was 100% for all spots. Gel spot
Accession no.
Systematic name
Protien name
Protien function
MW (Da)
pl
No. peptides matched
1 2 3
gij6323073 gij51013665 gij6324486
YLR044C YAL038W YOL086C
PdC1p Cdc19p Adh1p
Pyruvate decarboxylase Pyruvate kinase Alcohol dehydrogenase
61456.6 54909.5 36825.7
5.8 7.6 6.2
25 16 17
showed a slightly lower cell yield. Aiming to investigate if glutathione deficiency affected energy metabolism, we measured pyruvate kinase and aconitase activity and oxygen consumption in cells grown under calorie-restricted conditions (Fig. 3A). The results showed an increased activity of the glycolytic enzyme pyruvate kinase in calorie-restricted gsh1 mutant cells, suggesting that GSH deficiency favors a faster ATP production by fermentative growth. This would explain the increase in the cell yield of gsh1 mutant cells grown in 0.5% glucose medium. In agreement, aconitase activity and oxygen consumption were 40 and 21%
lower, respectively, in the gsh1 mutant (Fig. 3A and B). This decrease in mitochondrial function is consistent with the lower ROS production observed in aged gsh1 mutant cells, leading to an increased lifespan (Figs. 1A and 2A). Despite the involvement of GSH metabolism in the formation and maintenance of iron–sulfur clusters in mitochondrial enzymes (Sonq et al., 2006), the gsh1 null mutant strain showed a higher aconitase activity than WT when cells were grown under restricted respiratory conditions (data not shown). Therefore, the decrease in aconitase activity under CR reflects changes in metabolic fluxes in detriment of respiration.
Fig. 3. Glutathione deficiency reduces respiration rate of calorie-restricted cells. (A and B) Wild type (white bars) and gsh1 mutant cells (black bars) were grown under CR. Pyruvate kinase and aconitase activities (A) were measured in cells taken at t0 (before incubation at 37 8C), using protein extracts. Oxygen consumption (B) was measured in cell suspensions using a Clark electrode. Values are mean S.D. of three independent experiments. *p < 0.05.
704
S.C. Mannarino et al. / Mechanisms of Ageing and Development 129 (2008) 700–705
Fig. 4. Glutathione deficiency increases mutagenesis during chronological ageing. Wild type (white bars) and gsh1 mutant cells (black bars) were grown under CR, washed with water and incubated in water at 37 8C. (A) Mutagenic rate was measured as described in Section 2. (B) Competition assay. The same number of cells was taken from 72 haged cultures, mixed and incubated in fresh YPD 2% media. Cellular viability was followed at 0, 24 and 48 h. The percentage of viable cells of each strain was obtained by colony counting on YPD 2% and YPD 2% + geneticine plates. Values are mean S.D. of three independent experiments. **p < 0.01.
It was previously shown that respiration adapted yeast cells display an increased chronological lifespan, compared to cells pregrown on fermenting medium (MacLean et al., 2001; Harris et al., 2005; Piper et al., 2006). However, according to our results, CR gsh1 cells presented a decreased mitochondrial respiration but a longer chronological lifespan. To evaluate if this advantage for survival might only be a short-term benefit, we measured mitochondrial mutagenesis as the rate of petite formation during ageing. In cells aged for 6 h, the percentage of petites was similar to the observed at t0 (data not shown). However, as shown in Fig. 4A, GSH deficiency increased the mutation rate after 24 h of cell ageing, indicating that, although calorie-restricted gsh1 strain lives longer than WT, it accumulates mutations that are prejudicial for its population. We also investigated how aged cells adapted and recovered when inoculated in a fresh media. For both strains, cellular viability after 3 days of ageing are almost null, but when aged cells were reinoculated in fresh YPD 2% media, surviving cells were able to recover and grow. To analyze how these aged cultures compete for nutrients in the same environment, we mixed the same amount of WT and gsh1 mutant cells in fresh medium and monitored colonyforming units during recovery. To discriminate between the two strains, the mixed culture was plated on YPD 2% supplemented or not with geneticine (only gsh1 mutant cells are able to grow on both). As seen in Fig. 4B, WT strain dominated the environment, overwhelming the gsh1 strain. These results corroborate that GSH deficient cells have a short-term advantage but aged cells accumulate higher levels of mitochondrial DNA damage and are unable to compete against WT cells. 4. Discussion In this study, we showed that GSH deficiency decreased the metabolic switch from fermentation to respiration in CR cells. Nevertheless, chronological lifespan increased in CR gsh1 mutant cells. Under these conditions, the intracellular oxidation and protein carbonylation associated with the ageing process was decreased in gsh1 mutant cells. This was unexpected, since GSH is an important antioxidant molecule and lifespan is usually shortened in antioxidant deficient cells (Piper, 2006). The 2Danalysis showed that the carbonylation of pyruvate kinase, pyruvate decarboxylase and alcohol dehydrogenase 1 (Adh1), three proteins previously shown to be oxidized in aged cells (Reverter-Branchat et al., 2004), decreased in aged gsh1 mutant cells. Adh1 catalyses the conversion of acetaldehyde to ethanol, regenerating NAD+. It was previously shown that NAD+/NADH balancing mediated by this enzyme is important to chronological lifespan (Reverter-Branchat et al., 2007). The protection of Adh1 during ageing of gsh1 mutant cells may, therefore, contribute to lifespan extension.
The decrease in ROS production during ageing of gsh1 mutant cells was correlated with a decrease in aerobic metabolism, as indicated by a reduction in oxygen consumption and aconitase activity. In contrast, the activity of pyruvate kinase, one of the most controlled glycolytic enzyme, increased. Despite this short-term advantage, GSH deficiency increased the mitochondrial genome mutation rate during ageing, compromising the mutant at long term. This is consistent with the instability of the mitochondrial genome exhibited by gsh1 mutant cells (Lee et al., 2001). In agreement, aged WT cells outlasted aged gsh1 mutant cells in direct competition assays in a fresh medium. Since GSH deficiency decreased intracellular oxidation in aged cells, it seems unlikely that ROS mediate mitochondrial DNA damage. The carbonylation of pyruvate kinase, pyruvate decarboxylase and alcohol dehydrogenase in aged WT cells suggests that this strain may control its metabolism through inactivation of these proteins. Diverse published data shows that protein carbonylation is an irreversible modification that decreases protein activity. Therefore, protein carbonylation in aged WT cells impairs enzyme activity. As a consequence, the glycolytic flux would decrease and the desired respiration rate would be maintained to achieve a higher lifespan (compared to glucose repressed cells) and all the benefits that CR can provide. Although it had been reported that respiration is not required for the longevity benefits of CR in yeast (Kaeberlein et al., 2005), our results suggest that respiration confers long-term benefits of lifespan extension. Herker et al. (2004) showed that apoptosis in yeast confers a selective advantage for this organism, by eliminating defective cells from culture and saving nutrients for the remaining cells. When yca1 null mutants, deficient in the yeast metacaspase, have to compete against WT cells, the latter show better survivals in the end. Recently it was shown by Gomes et al. (2008) that a glutaredoxin system, which is dependent on GSH, is involved in apoptosis signaling under cadmium stress. Whether apoptosis signaling is compromised in the gsh1 mutant strain, affecting its ability to remove undesired or damaged cells during the ageing process, is an important issue to be investigated in further studies. In summary, our results show that glutathione deficiency increases chronological lifespan of CR cells. This lifespan extension is correlated with lower levels of oxidative stress markers, including a decrease in ROS and Adh1 carbonylation that was associated with a reduction in respiration metabolism. However, gsh1 null mutants accumulate high levels of mitochondrial DNA damage during cell ageing and are unable to compete with aged WT cells. In the WT strain, despite the higher respiration rate and the more oxidized intracellular environment in aged cells, cells are able to prevent mutations and are fit to compete with gsh1 mutant strain, which guarantee a long-term advantage.
S.C. Mannarino et al. / Mechanisms of Ageing and Development 129 (2008) 700–705
Acknowledgements We thank Prof. Reginaldo Menezes for expert assistance in oxygen consumption analyses. This project was supported by CAPES (GRICES/PROCAD), CNPq, FAPERJ and FAPESP (grant 0410067/6). References Adamis, P.D., Gomes, D.S., Pinto, M.L., Panek, A.D., Eleutherio, E.C., 2004. The role of glutathione transferases in cadmium stress. Toxicol. Lett. 154, 81–88. Adamis, P.D., Panek, A.D., Eleutherio, E.C., 2007. Vacuolar compartmentation of the cadmium–glutathione complex protects Saccharomyces cerevisiae from mutagenesis. Toxicol. Lett. 173, 1–7. Balaban, R.S., Nemoto, S., Finkel, T., 2005. Mitochondria, oxidants and aging. Cell 120, 483–495. Barja, G., 2002. Endogenous oxidative stress: relationship to aging, longevity and caloric restriction. Ageing Res. Rev. 1, 397–411. Barros, M.H., Bandy, B., Tahara, E.B., Kowaltowski, A.J., 2004. Higher respiratory activity decreases mitochondrial reactive oxygen release and increases life span in Saccharomyces cerevisiae. J. Biol. Chem. 279, 49883–49888. Bergmeyer, H.U., 1974. Methods of Enzymatic Analysis, 2nd ed., vol. I. Academic Press, pp. 465. Bitterman, K.J., Medvedik, O., Sinclair, D.A., 2003. Longevity regulation in Saccharomyces cerevisiae: linking metabolism, genome stability, and heterochromatin. Microbiol. Mol. Biol. Rev. 67, 376–399. Bonawitz, N.D., Rodeheffer, M.S., Shadel, G.S., 2006. Defective mitochondrial gene expression results in reactive oxygen species-mediated inhibition of respiration and reduction of yeast life span. Mol. Cell. Biol. 26, 4818–4829. Brennan, R.J., Schiestl, R.H., 1996. Cadmium is an inducer of oxidative stress in yeast. Mutat. Res. 356, 171–178. Chouchane, S., Snow, E., 2001. In vitro effect of arsenical compounds on glutathionerelated enzymes. Chem. Res. Toxicol. 14, 517–522. Costa, V., Amorim, M.A., Quintanilha, A., Moradas-Ferreira, P., 2002. Hydrogen peroxide-induced carbonylation of key metabolic enzymes in Saccharomyces cerevisiae: the involvement of the oxidative stress response regulators Yap1 and Skn7. Free Radic. Biol. Med. 33, 1507–1515. Davidson, J.F., Whyte, B., Bissinger, P.H., Schiestl, R.H., 1996. Oxidative stress is involved in heat-induced death in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. U.S.A. 93, 5116–5121. Dillin, A., Hsu, A.L., Arantes-Oliveira, N., Lehrer-Graiwer, J., Hsin, H., Fraser, A.G., Kamath, R.S., Ahringer, J., Kenyon, C., 2002. Rates of behavior and aging specified by mitochondrial function during development. Science 298, 2398–2401. Fabrizio, P., Gattazzo, C., Battistella, L., Wei, M., Cheng, C., 2005. Sir2 blocks extreme life-span extension. Cell 123, 655–667. Fabrizio, P., Liou, L.L., Moy, V.N., Diaspro, A., Valentine, J., 2003. SOD2 functions downstream of Sch9 to extend longevity in yeast. Genetics 163, 35–46. Fabrizio, P., Longo, V.D., 2003. The chronological life span of Saccharomyces cerevisiae. Aging Cell 2, 73–81. Feng, J., Bussiere, F., Hekimi, S., 2001. Mitochondrial electron transport is a key determinant of life span in Caenorhabditis elegans. Dev. Cell 1, 633–644. Finehout, E.J., Franck, Z., Lee, K.H., 2004. Towards two-dimensional electrophoresis mapping of the cerebrospinal fluid proteome from a single individual. Electrophoresis 25, 2564–2575. Gardner, P.R., Raineri, I., Epstein, L.B., White, C.W., 1995. Superoxide radical and iron modulate aconitase activity in mammalian cells. J. Biol. Chem. 270, 13399– 13405. Glock, G.E., McLean, P., 1953. Further studies on the properties and assay of glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase of rat liver. Biochem. J. 55, 400–408. Gomes, D.S., Fragoso, L.C., Riger, C.J., Panek, A.D., Eleutherio, E.C., 2002. Regulation of cadmium uptake by Saccharomyces cerevisiae. Biochim. Biophys. Acta 1573, 21– 25. Gomes, D.S., Pereira, M.D., Panek, A.D., Andrade, L.R., Eleutherio, E.C., 2008. Apoptosis as a mechanism for removal of mutated cells of Saccharomyces cerevisiae: the role of Grx2 under cadmium exposure. Biochim. Biophys. Acta 1780, 160–166. Gunasekaran, S., Imbayagwo, M., McDonald, L., 1995. Influence of carbon and nitrogen sources on glutathione catabolic enzymes in Candida albicans during dimorphism. Mycopathologia 131, 93–97. Harris, N., Costa, V., MacLean, M., Mollapour, M., Moradas-Ferreira, P., Piper, P.W., 2003. Mn-Sod overexpression extends the yeast chronological (G0) life span, but acts independently of Sir2p histone deacetylase to shorten the replicative life span of dividing cells. Free Radic. Biol. Med. 34, 1599–1606.
705
Harris, N., Bachler, M., Costa, V., Mollapour, M., Moradas-Ferreira, P., Piper, P.W., 2005. Overexpressed Sod1p acts either to reduce or to increase the lifespans and stress resistance of yeast, depending on whether it is Cu-deficient or an active Cu, Zn-superoxide dismutase. Aging Cell 4, 41–52. Herker, E., Jungwirth, H., Lehmann, K.A., Maldener, C., Fro¨hlich, K.U., Wissing, S., Bu¨ttner, S., Fehr, M., Sigrist, S., Madeo, F., 2004. Chronological aging leads to apoptosis in yeast. J. Cell Biol. 164, 501–507. Jazwinski, S.M., 1999. Longevity, genes, and aging: a view provided by a genetic model system. Exp. Gerontol. 34, 1–6. Jiang, J.C., Jaruga, E., Repnevskaya, M.V., Jazwinski, S.M., 2000. An intervention resembling caloric restriction prolongs life span and retards aging in yeast. FASEB J. 14, 2135–2137. Kaeberlein, M., Burtner, C.R., Kennedy, B.K., 2007. Recent developments in yeast aging. PLoS Genet. 3, e84. Kaeberlein, M., Hu, D., Kerr, E.O., Tsuchiya, M., Westman, E.A., 2005. Increased life span due to calorie restriction in respiratory-deficient yeast. PLoS Genet. 1, e69. Lee, J.C., Straffon, M.J., Jang, T.Y., Higgins, V.J., Grant, C.M., Dawes, I.W., 2001. The essential and ancillary role of glutathione in Saccharomyces cerevisiae analysed using a grande gsh1 disruptant strain. FEMS Yeast Res. 1, 57–65. Lin, S.J., Defossez, P.A., Guarente, L., 2000. Requirement of NAD and SIR2 for life-span extension by calorie restriction in Saccharomyces cerevisiae. Science 289, 2126– 2128. Lin, S.J., Kaeberlein, M., Andalis, A.A., Sturtz, L.A., Defossez, P.A., Culotta, V.C., Fink, G.R., Guarente, L., 2002. Calorie retriction extends Saccharomyces cerevisiae lifespan by increasing respiration. Nature 418, 344–348. Lin, S.J., Ford, E., Haigis, M., Liszt, G., Guarente, L., 2004. Calorie restriction extends yeast life span by lowering the level of NADH. Genes Dev. 18, 12–16. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, L.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275. MacLean, M.J., Harris, N., Piper, P.W., 2001. Chronological life span of stationary phase yeast cells: a model for investigating the factors that might influence the ageing of postmitotic tissues in higher organisms. Yeast 18, 499–509. Merry, B.J., 2004. Oxidative stress and mitochondrial function with aging—the effects of calorie restriction. Aging Cell 3, 7–12. Penninckx, M.J., 2002. An overview of glutathione in Saccharomyces versus nonconventional yeasts. FEMS Yeast Res. 2, 295–305. Pereira, M.D., Herdeiro, R.S., Fernandes, P.N., Eleutherio, E.C.A., Panek, A.D., 2003. Targets of oxidative stress in yeast sod mutants. Biochim. Biophys. Acta 1620, 245–251. Piper, P.W., 2006. Long-lived yeast as a model for ageing research. Yeast 23, 215– 226. Piper, P.W., Harris, N.L., MacLean, M., 2006. Preadaptation to efficient respiratory maintenance is essential both for maximal longevity and the retention of replicative potential in chronologically aging yeast. Mech. Ageing Dev. 127, 733–740. Powers III, R.W., Kaeberlein, M., Caldwell, S.D., Kennedy, B.K., Fields, S., 2006. Extension of chronological life span in yeast by decreased TOR pathway signaling. Genes Dev. 20, 174–184. Reverter-Branchat, G., Cabiscol, E., Tamarit, J., Ros, J., 2004. Oxidative damage to specific proteins in replicative and chronological-aged Saccharomyces cerevisiae: common targets and prevention by calorie restriction. J. Biol. Chem. 279, 31983–31989. Reverter-Branchat, G., Cabiscol, E., Tamarit, J., Sorolla, M.A., Torre, M.A., Ros, J., 2007. Chronological and replicative life-span extension in Saccharomyces cerevisiae by increased dosage of alcohol dehydrogenase 1. Microbiology 153, 3667–3676. Speakman, J.R., Talbot, D.A., Selman, C., Snart, S., McLaren, J.S., Redman, P., Krol, E., Jackson, D.M., Johnson, M.S., Brand, M.D., 2004. Uncoupled and surviving: individual mice with high metabolism have greater mitochondrial uncoupling and live longer. Aging Cell 3, 87–95. Sohal, R.S., Weindruch, R., 1996. Oxidative stress, caloric restriction, and aging. Science 273, 59–63. Sohal, R.S., 2002. Oxidative stress hypothesis of aging. Free Radic. Biol. Med. 33, 573–574. Sonq, J.Y., Cha, J., Lee, J., Roe, J.H., 2006. Glutathione reductase and a mitochondrial thioredoxin play overlapping roles in maintaining iron–sulfur enzymes in fission yeast. Eukaryot. Cell 5, 1857–1865. Stickland, L.H., 1951. The determination of small quantities of bacteria by means of the Biuret reaction. J. Gen. Microbiol. 5, 698–703. Sto¨ckl, P., Zankl, C., Hu¨tter, E., Unterluggauer, H., Laun, P., Heeren, G., Bogengruber, E., Herndler-Brandstetter, D., Breitenbach, M., Jansen-Du¨rr, P., 2007. Partial uncoupling of oxidative phosphorylation induces premature senescence in human fibroblasts and yeast mother cells. Free Radic. Biol. Med. 43, 947–958. Thomas, J.A., Mallis, R.J., 2001. Aging and oxidation of reactive protein sulfhydryls. Exp. Gerontol. 36, 1519–1526.