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1 IBMC, Instituto de Biologia Molecular e Celular, Grupo de Microbiologia Celular e Aplicada, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal
2 ICBAS, Instituto de Ciências Biomédicas Abel Salazar, Departamento de Biologia Molecular, Universidade do Porto, Porto, Portugal
3 Department of Cell and Molecular Biology, Göteborg University, Box 462, S-405 30 Göteborg, Sweden
Correspondence
Vítor Costa
vcosta{at}ibmc.up.pt
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The data from this study have been deposited in the microarray data public repository ArrayExpress under the accession number E-MEXP-326.
A summary of genes differentially expressed during the recovery of yeast cells from H2O2 stress is available as supplementary data with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). When the level of ROS exceeds the antioxidant capacity, redox homeostasis is disrupted, and molecules such as nucleic acids, lipids, proteins and carbohydrates are oxidized. Studies using yeast cells have contributed to the characterization of cellular functions involved in protection against oxidative stress (Jamieson, 1998; Moradas-Ferreira & Costa, 2000; Thorpe et al., 2004), and to the identification of the major protein targets oxidatively inactivated (Cabiscol et al., 2000; Costa et al., 2002; Reverter-Branchat et al., 2004). Protein oxidative modifications include the reversible oxidation of sulphur-containing amino acids, and the irreversible conversion of some amino acid residues to carbonyl derivatives. The oxidation of protein cysteine residues generates sulphenic acid derivatives that are S-thiolated with reduced glutathione (Grant et al., 1999; Demasi et al., 2003; Shenton & Grant, 2003), or further oxidized to sulphinic acid moieties. Protein activity is restored by the monothiol glutaredoxin Grx5p (Shenton et al., 2002) or by sulphiredoxin (Biteau et al., 2003), respectively. Protein methionine residues are oxidized to methionine sulphoxide, which can be reduced by methionine sulphoxide reductase (Moskovitz et al., 1997). The accumulation of oxidized proteins has been implicated in the initiation and progression of numerous diseases and ageing (Petropoulos & Friguet, 2005). To prevent the accumulation of irreversibly oxidized proteins, cells have to respond by increasing the rate of proteolysis (Dunlop, 2002). Therefore, protein degradation, in addition to repair, plays a key housekeeping role for eliminating oxidized proteins.
Proteins can be degraded by the proteasome or by vacuolar/lysosomal proteases. The 26S proteasome is assembled by association of the 20S proteasome catalytic core with the 19S regulatory particle. A number of studies have indicated that the 20S proteasome, which degrades proteins in an ATP- and ubiquitin-independent manner, is responsible for the degradation of oxidized proteins. Indeed, ubiquitin-activating and -conjugating enzymes and the 26S proteasome are inhibited during oxidative stress, and cells deficient in ubiquitin-conjugating activity are able to degrade oxidized proteins at near normal rates (Shringarpure et al., 2003). In yeast, exposure to H2O2 increases the activity of the 20S proteasome. Furthermore, cells deficient in the Rpn9p subunit of the 19S regulatory complex exhibit a higher activity of the 20S proteasome, and are able to degrade carbonylated proteins more efficiently than are wild-type cells (Inai & Nishikimi, 2002). The degradation of cytosolic components by lysosomes (autophagy) also plays an essential role in the maintenance of cellular homeostasis (Massey et al., 2004). In mammalian cells, it has recently been shown that a chaperone-mediated autophagy pathway is activated by oxidative stress, and is important for the efficient removal of oxidized cytosolic proteins by lysosomes (Kiffin et al., 2004).
The progressive accumulation of malfunctioning cell components during chronological ageing of post-mitotic cells is associated with oxidative damage (Reverter-Branchat et al., 2004; Petropoulos & Friguet, 2005; Vijg & Suh, 2005; Harris et al., 2005) and age-dependent decline of turnover rate and housekeeping (Chen et al., 2004). Consistently, the deletion of UMP1, a gene necessary for 20S proteasome biogenesis, increases constitutive and ageing-induced protein oxidation in yeast (Chen et al., 2004, 2005). The decrease in the activity of both the proteasomal system and the lysosomal proteases, accompanied by an increased accumulation of oxidized proteins, has been observed during senescence of non-dividing fibroblasts (Grune et al., 2005).
In this study, we show that protein fate is a significantly over-represented function induced during recovery of yeast cells after H2O2 stress. In addition to genes encoding subunits of the 20S proteasome, the PEP4 and LAP4 genes, encoding vacuolar proteases, as well as genes associated with protein sorting into the vacuole and vacuolar fusion, were upregulated. Protein turnover increased after oxidative damage by a mechanism partially dependent on Pep4p, which is required for the post-translational precursor maturation of vacuolar proteinases (Woolford et al., 1986). In agreement, the degradation of carbonylated proteins decreased in cells lacking Pep4p. The key role of vacuolar proteolysis in the removal of oxidized cytosolic proteins was further supported by data showing that Pep4p activity increased during chronological ageing, and pep4
mutants showed premature senescence associated with increased accumulation of carbonylated proteins. However, the increased removal of oxidized proteins by PEP4 overexpression was not sufficient to enhance chronological lifespan.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, ade2-1, can1-100, trp1-1, ura3-1, his3-11,15, leu2-3,112) (Wallis et al., 1989), W303 pep4 : : HIS3 (this work), W303 pDP34 (this work), W303 pDP34-PEP4 (this work) and W303 p416ADH-PEP4-GFP (this work). For PEP4 (=PRA1) disruption, a 3.0 kb deletion fragment containing HIS3 and the flanking regions of PEP4 was used. This fragment was obtained from pKS-pra1EN : : HIS3 (Hirsch et al., 1992), using the following specific primers: reverse (5'-ACACAGGAAACAGCTATGAC-3') and forward (5'-AGGGTTTTCCCAGTCACGAC-3'). Yeast cells were transformed by electroporation. Gene disruption was confirmed by PCR analysis. Yeast cells were grown in YPD [1 % (w/v) yeast extract, 2 % (w/v) bactopeptone, 2 % (w/v) glucose] or in minimal medium [0.67 % (w/v) yeast nitrogen base without amino acids, 2 % (w/v) glucose], supplemented with appropriate amino acids (40 mg tryptophan l–1, 40 mg histidine l–1, 80 mg leucine l–1, 40 mg methionine l–1) and nucleotides (40 mg adenine l–1, 40 mg uracil l–1), to early exponential phase (OD600 0.6) or post-diauxic phase (OD600 7–8 in YPD; OD600 4 in minimal medium), in an orbital shaker, at 26 °C and 120 r.p.m.
H2O2 recovery and chronological lifespan assay.
In H2O2 recovery experiments, yeast cells were grown in minimal medium to the exponential phase, treated with 1.5 mM H2O2 for 30 min, and centrifuged at 4500 r.p.m. for 5 min. Yeast cells were resuspended in minimal medium lacking H2O2, and allowed to recover for the indicated times. Chronological lifespan was assayed as previously described (Harris et al., 2005). Yeast cells were grown to the post-diauxic phase, centrifuged at 4000 r.p.m. for 5 min, and washed twice with water. Cells were resuspended in water, and incubated at the indicated temperature. Cell viability was determined by standard dilution plate counts on YPD medium containing 1.5 % agar. Colonies were counted after growth at 26 °C for 3 days.
Glucose assay and enzyme activities.
Glucose in the growth medium was quantified by the glucose/peroxidase method. Samples (2 ml) of cultures were taken and centrifuged at 4500 r.p.m. for 5 min, and supernatants were stored at –20 °C until use. The TGO reagent [0.5 M Tris, pH 7.0, 20 U glucose oxidase ml–1, 0.38 U peroxidase ml–1, 0.05 mg o-dianisidine hydrochloride ml–1, 1 % (v/v) Triton X-100] (2.5 ml) was added to 0.5 ml of sample; the mixture was incubated for 20 min at 37 °C, and the absorbance at 420 nm was measured. Glucose was estimated by reference to a standard curve prepared with known amounts of glucose. For the proteinase A (Pep4p) activity assay, yeast extracts were prepared in 0.1 M Tris, pH 7.5, by vigorous shaking of the cell suspension, in the presence of glass beads, for 5 min. Short pulses of 1 min were used, at 1 min intervals, on ice. Pep4p activity was determined using 0.5 mg total protein, by measuring the release of tyrosine-containing acid-soluble peptides from acid-denatured haemoglobin [expressed as µg Tyr min–1 (mg protein)–1] (Jones, 1990). Glyceraldehyde-3-phosphate dehydrogenase activity (Tdh) was quantified as described by Holland & Westhead (1973), using 25 µg total protein, and expressed as U (mg protein)–1, or as percentage of the control. Protein content of cellular extracts was estimated by the Lowry method, using BSA as a standard.
Pep4–GFP levels.
Proteins were isolated from S. cerevisiae W303 p416-PEP4–GFP cells, as described for Pep4 activity, and separated by native PAGE (10 % gel). After electrophoresis, Pep4–GFP was detected using a molecular imager (Typhoon; Amersham Biosciences Europe). For loading control, proteins were stained with 0.25 % (w/v) Coomassie blue R250, 10 % (v/v) acetic acid/45 % (v/v) methanol, and destained with 10 % acetic acid/45 % methanol.
mRNA preparation, synthesis of cDNA, Genefilters hybridization and data analysis.
Total RNA was isolated by the acid–phenol method (Ausubel et al., 1998). Genefilters yeast microarrays (Research Genetics) were hybridized using [33P]CTP-labelled cDNA, as described previously (Rep et al., 2000). A Molecular Imager FX (Bio-Rad) was used to obtain a digital image of the filters. Images were converted to tagged image file format (TIFF), and the Pathways4 software (Research Genetics) was used for quantification of spot intensities, and for pairwise comparison of gene filter images. Prior to determination of changes in gene expression, all spot intensities were normalized by dividing sampled intensities by the mean sampled intensities of all clones. To determine the fold induction or repression, the relative mRNA level was expressed as the ratio H2O2/control (untreated) or recovery/H2O2. Genes that changed at least twofold were considered for further analysis. All values are means of the expression profiles of four experiments with similar results, using independent cultures grown or treated under the same conditions. Statistical analysis of the microarray data was performed by using BRB ArrayTools (version 3.3.0 beta1) developed by Richard Simon and Amy Peng Lam (http://linus.nci.nih.gov/BRB-ArrayTools.html). The data were deposited in the microarray data public repository ArrayExpress (http://www.ebi.ac.uk/arrayexpress/) (Parkinson et al., 2005) under the accession number E-MEXP-326. Statistical analysis of over-representation of functional groups was performed by using FUNSPEC (Robinson et al., 2002). All available databases were addressed by using a probability cut-off of 0.01 and the Bonferroni correction for multiple testing.
Northern blotting.
Total RNA (30 µg) was denaturated with glyoxal and DMSO, blotted onto Hybond-N membranes, and probed, as described by Ausubel et al. (1998). The following probes were used: a 1 kb HindIII–EcoRI fragment of the ACT1 gene, and a 787 bp fragment of the PEP4 gene amplified by PCR, using PRA1F (5'-GGGAGCATCCTTTCTTCACTG-3') and PRA1R (5'-TGCAGAGATACAGGAGCCTGA-3') primers. Probes were labelled with [32]P-dCTP using the Multiprime DNA Labelling System (RPN 1601Z; Amersham Biosciences Europe). Band intensities were evaluated by densitometry.
Pulse–chase.
Yeast cells (5 ml per sample) were incubated with 3.7x105 Bq [35S]methionine ml–1 for 30 min, centrifuged and resuspended in 5 ml minimal medium supplemented with methionine. 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), by vigorous shaking of the cell suspension, in the presence of glass beads, for 5 min. Short pulses of 1 min were used, at 1 min intervals, on ice. Proteins (500 000 c.p.m.) were separated by SDS-PAGE (12.5 % gel). Gels were dried and exposed to an X-ray film. Band intensities were quantified by densitometry.
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), as described for Pep4 activity. For 1D analysis, proteins (15 µg) were derivatized with 2,4-dinitrophenylhydrazine (DNPH) (Levine et al., 1994), and separated by SDS-PAGE (12.5 % polyacrylamide gel). After electrophoresis, proteins were stained with Coomassie blue, or blotted onto Hybond-ECL membranes (Amersham Biosciences Europe). Slot blotting was performed using PVDF membranes (Hybond-PVDF; Amersham Biosciences Europe), as previously described (Costa et al., 2002). The ECL or PVDF membranes were probed with rabbit IgG anti-DNP (Dako) at a 1 : 5000 dilution as the primary antibody, and goat anti-rabbit IgG–peroxidase (Sigma) at a 1 : 5000 dilution as the secondary antibody. Immunodetection was performed by chemiluminescence, using a kit from Amersham (RPN 2109). The membranes were exposed to a Hybond-ECL film (Amersham Biosciences Europe) for 15 s to 1 min, and the film was developed. Band intensities were quantified by densitometry.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Lee et al., 1999; Gasch et al., 2000). During cellular recovery after H2O2 stress (recovery versus H2O2) the mRNA level of 99 genes increased, whereas that of another 169 genes (including 88 encoding ribosomal proteins) was diminished (see Supplementary Table S1). Genes up- or down-regulated during recovery that were repressed or induced, respectively, during previous exposure to H2O2 were not considered to be specific, because these changes merely restored control mRNA levels. Genes differentially expressed were sorted into functional categories according to the Munich Information Center for Protein Sequences (MIPS). Our data showed that some categories were significantly more represented in cells after recovery, compared to those in H2O2-treated cells (Fig. 2): 57 % of the genes down-regulated were associated with protein biosynthesis, whereas 32 % of the upregulated genes were related to protein fate, and 37 % to metabolism. To identify over-represented biological processes, gene lists were analysed by using the FUNSPEC software (Robinson et al., 2002). Among induced genes, protein catabolism functions had the greatest significance (Table 1). The induction (two- to threefold) of proteolytic activity was correlated with the upregulation of amino acid catabolism, and the down-regulation of amino acid biosynthesis. Genes encoding subunits of the 20S proteasome, as well as some associated with ubiquitin-dependent proteolysis, were found to be upregulated. These results are consistent with published data showing that the 20S proteasome is required for degradation of oxidized proteins (Shringarpure et al., 2003: Inai & Nishikimi, 2002; Chen et al., 2004, 2005).
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). Lap4p is a leucine aminopeptidase that is transported into the vacuole via an autophagosome (Suzuki et al., 2002). Interestingly, genes associated with protein sorting into the vacuole, and with vacuolar fusion (MVP1, 3.3-fold; YHR138C, 2.5-fold; see Supplementary Table S1), were also upregulated.
To confirm the induction of PEP4 gene expression, mRNA-PEP4, Pep4p activity and Pep4p levels were analysed in cells allowed to recover for up to 4 h. The results obtained showed that mRNA-PEP4 and Pep4p protein levels increased during the first 2 h of recovery (Fig. 3a, b). In agreement, Pep4p activity increased twofold (Fig. 3b). After 4 h of recovery, mRNA-PEP4 returned to control levels; however, Pep4p levels and enzyme activity remained high. This transient upregulation of PEP4 gene expression, leading to a more sustained increase in protein and enzyme activity, suggests that Pep4p is stable and has a long half-life.
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). In control cells (untreated), protein degradation was not observed at up to 4 h of recovery (data not shown).
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). In cycloheximide-treated cells, Tdh activity increased to <60 % of control levels (Fig. 5). Interestingly, Tdh activity after cell recovery was similar to that achieved by in vitro dethiolation. These results indicate that de novo protein synthesis contributes to restoration of the active enzyme.
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). As PEP4 and LAP4 genes were induced, and Pep4p activity increased during recovery from H2O2 stress, we raised the hypothesis that vacuolar proteolysis contributes to protein turnover after oxidative damage. To test this hypothesis, the PEP4 gene was disrupted by homologous recombination, and the rate of protein degradation in pep4 mutants was compared with that of parental cells. Notably, loss of Pep4p significantly decreased protein degradation during cellular recovery (Fig. 4a
). The correlation between protein turnover and removal of oxidized proteins was further analysed by measuring protein carbonyl content. Consistent with earlier studies (Inai & Nishikimi 2002), protein carbonylation increased during exposure of parental cells to 1.5 mM H2O2, and decreased as cells were allowed to recover (Fig. 4b). The deficiency in Pep4p led to an increase in the basal levels of protein carbonyl groups. The analysis of carbonyl content by slot blot analysis confirmed this increase (146±14 %, pep4 versus W303, P<0.001; data not shown). Furthermore, although H2O2-induced protein carbonylation in the pep4 mutants was of the same order of magnitude as that of the parental strain, the decrease in carbonyl content during cellular recovery occurred at a much lower rate (Fig. 4b).
The vacuolar protease Pep4p is required for the removal of oxidized proteins during chronological ageing, but PEP4 overexpression is not sufficient to increase lifespan
The progressive accumulation of misfunctioning cell components due to oxidative damage is associated with ageing. In yeast cells, chronological lifespan has been studied by measuring the capacity of post-mitotic cells (growth-arrested in water) to maintain viability over time. Under these conditions (nutrient starvation), intracellular vacuolar catabolism is activated, and protein-based nitrogen recycling is important for cell survival (Teichert et al., 1989; Levine & Klionsky, 2004). To investigate if the homeostatic function of the vacuole is important for the turnover of oxidized proteins during chronological ageing, yeast cells were grown in YPD medium to the post-diauxic (respiratory) phase, growth-arrested in the G0 phase, and incubated at 37 °C. The results showed that parental cells started to lose viability after 15 days of ageing (Fig. 6a). As previously shown (Harris et al., 2005), cell ageing was correlated with protein oxidation: the protein carbonyl content increased two- and sixfold in yeast cells aged for 7 and 15 days, respectively (Fig. 6b). This dramatic increase in total protein carbonyl content preceded a rapid decline in cell viability between the 15- and 21-day time points. Yeast cells lacking the Pep4p proteinase displayed premature ageing associated with increased accumulation of protein carbonyl groups (Fig. 6a, b): at day 2, 100 % of parental cells remained viable, and protein carbonyl content was similar to that in the controls; and in pep4 mutants, only 3 % of the cells remained viable, and carbonyl content increased 3.4-fold. The accumulation of oxidized proteins has also been linked to an age-dependent decline in turnover rate and housekeeping (Chen et al., 2004). Therefore, Pep4p activity was determined during chronological ageing. Our data showed an age-dependent increase in Pep4p activity, and did not reveal a decrease when cells started to lose viability (Fig. 6c), suggesting that Pep4p activation was not sufficient to counteract the protein oxidation rate.
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), and grown in minimal medium to the post-diauxic phase. Enzyme activity increased 5.7-fold in PEP4 cells (data not shown). The lifespan of cells previously grown in minimal medium was significantly reduced relative to that in cells grown in rich medium (data not shown). Therefore, chronological lifespan was measured at the less stressful temperature of 30 °C. As shown in Fig. 7, PEP4 overexpression did not increase lifespan, although it completely prevented the increase in protein oxidation associated with cell ageing. The overall results suggest that the vacuolar protease Pep4p is important for maintaining cellular homeostasis by degrading oxidized proteins, in addition to its well-characterized function of supplying nitrogen under nutritional stress conditions (Teichert et al., 1989). However, increased recycling of damaged proteins was not sufficient to extend lifespan. This may be explained by the fact that PEP4 overexpression does not prevent the accumulation of other oxidatively modified molecules (lipids and DNA) that is associated with cell death.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Costa et al., 2002). Supporting the hypothesis that protein turnover is important for cellular recovery after oxidative damage, we showed that de novo protein synthesis contributes to restoring Tdh activity.
Several studies have indicated that the 20S proteasome plays a key role in the removal of irreversibly oxidized proteins (Shringarpure et al., 2003; Inai & Nishikimi, 2002). In agreement, the expression of several genes encoding 20S proteasome subunits (PRE3, PRE6, PRE7, PRE9 and UMP1) increased during recovery. Although we also observed the induction of genes encoding ubiquitin-activation (UBA1) and -conjugation (UBC1, RAD6) enzymes, a ubiquitin-specific protease (UBP6) and one 26S proteasome subunit (UBC13), the degradation of carbonylated proteins does not require ubiquitination. Indeed, cells deficient in Doa4p, which contain reduced levels of free ubiquitin (Swaminathan et al., 1999), degrade carbonylated proteins as efficiently as do parental cells (data not shown), and protein multi-ubiquitination is not observed in H2O2-treated cells (Inai & Nishikimi, 2002).
Our data show that genes associated with vacuolar proteolysis are also upregulated during recovery after oxidative damage. These include PEP4, which encodes a vacuolar aspartyl protease required for the post-translational precursor maturation of vacuolar proteinases (Woolford et al., 1986), and LAP4, which encodes a vacuolar aminopeptidase, as well as genes related to protein sorting into the vacuole, and to vacuolar fusion. Consistent with a role for vacuolar proteolysis in cellular recovery from oxidative damage, Pep4p levels and enzyme activity increased during recovery, and this induction was correlated with a higher rate of proteolysis, and a decrease in protein carbonyl content. In addition, the protein degradation rate and the capacity to remove oxidized proteins decreased in pep4 mutants. Importantly, the oxidized protein level was constitutively higher in pep4 mutants, indicating that vacuolar proteolysis has a major role in the turnover of proteins oxidized by endogenously generated or exogenously added ROS. These results are in agreement with data suggesting that protein sorting, vacuole function and vacuolar acidification are core functions required for broad resistance to oxidative stress in yeast (Thorpe et al., 2004). In yeast, a mechanism of transport into the vacuole independent of the secretory pathway has been described for a few cytosolic proteins, namely the gluconeogenic enzyme fructose-1,6-bisphosphatase (Brown et al., 2000) and the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (Horst et al., 1999). Members of the Hsp70 family play a role in the import of these cytosolic proteins into the vacuole. In mammalian cells, it has recently been shown that a chaperone-mediated autophagy pathway is activated by oxidative stress, and contributes to degradation of oxidized cytoplasmic proteins inside the lysosomes (Kiffin et al., 2004). The present study identified several genes associated with protein folding and stabilization that are upregulated during recovery after H2O2 stress, and therefore, might be important for the selective import of oxidized proteins into the vacuole. However, it has recently been shown that cells treated with H2O2 release Pep4p into the cytoplasm (Mason et al., 2005). It is therefore conceivable that oxidized proteins may be degraded by Pep4p in the cytoplasm.
Studies in yeast and other model organisms have shown that the increased scavenging of ROS by overexpression of antioxidant defences delays ageing (Sun et al., 2002; Harris et al., 2003, 2005), and defective removal of damaged proteins by the 20S proteasome promotes senescence (Chen et al., 2004, 2005). The key role of Pep4p in the turnover of damaged proteins after oxidative stress led us to investigate if this homeostatic function is important for chronological lifespan. It has been shown that Pep4p is important for survival of stationary cells, as it recycles nitrogen in starved cells (Teichert et al., 1989; Levine & Klionsky, 2004). The present study showed that the premature senescence of pep4 mutants was associated with increased accumulation of carbonylated proteins. It should be noted that protein carbonyl content in the parent strain at day 15, when cells began to lose viability, was 1.8-fold higher than the amount in aged pep4 mutants (day 2) (Fig. 6b). The dramatic shortening of the chronological lifespan in cells lacking Pep4p was probably due to the sum of two detrimental effects: nitrogen starvation and accumulation of damaged proteins.
In parental cells, Pep4p activity increased during cell ageing, indicating that lifespan was not limited by the loss of Pep4p function. Thus, protein oxidation rate may exceed the capacity of aged cells to degrade oxidized proteins in the vacuole. A progressive decline in the capacity to deliver cargo into the vacuole would also explain these results. Interestingly, orthologues of yeast autophagy genes are essential for lifespan extension by calorie restriction, and for the increased longevity of Caenorhabditis elegans mutants with defective Daf-2 protein, an insulin receptor analogue (Melendez et al., 2003).
The steady accumulation of carbonylated proteins over time was prevented in cells overexpressing PEP4, supporting the role of Pep4p in the turnover of oxidized proteins. Despite this, the increased Pep4p activity did not extend yeast chronological lifespan. These results indicate that removal of oxidized proteins is not the (only) limiting factor in lifespan. This may be explained by the fact that increased levels of Pep4p do not prevent the increase of other hallmarks of cell ageing, such as lipid peroxidation and oxidative DNA damage (Halliwell & Gutteridge, 1999). In contrast, overexpression of free-radical-scavenging enzymes (primary defences) decreases intracellular oxidation, preventing oxidative damage to cellular molecules, and therefore extending lifespan (Sun et al., 2002; Harris et al., 2003, 2005). Nevertheless, this does not exclude the hypothesis that the accumulation of oxidized proteins promotes cell death associated with ageing.
In summary, the overall results indicate that vacuolar proteolysis contributes to the efficient removal of proteins oxidized during exposure to H2O2, or by ROS generated as by-products of normal aerobic metabolism. This work also provides new evidence that failure to complete housekeeping due to defective vacuolar proteolysis contributes to the progressive accumulation of molecular damage associated with chronological ageing. However, removal of oxidized proteins is not the only limiting factor, as PEP4 overexpression prevents the accumulation of carbonylated proteins, but is not sufficient to extend lifespan.
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ACKNOWLEDGEMENTS |
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EN : : HIS3, pDP34 and pDP34-PEP4, and Professor D. S. Goldfarb (University of Rochester) for p416ADH-PEP4-GFP. |
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Received 4 April 2006;
revised 31 July 2006;
accepted 21 August 2006.
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0.6). Control (
) and cells treated with 1.5 mM H2O2 for 30 min (
) were centrifuged and resuspended in minimal medium without H2O2. Cell density (a) and percentage (w/v) glucose remaining in the growth medium (b) were determined during cellular recovery up to 24 h. Values are means±SD of three independent experiments.
) cells by measuring cell viability on the days indicated. (b) Carbonyl content in W303 (open bars) and pep4