RESEARCH ARTICLE

CTT1 overexpression increases life span of calorie-restrictedSaccharomyces cerevisiae deficient in Sod1

Germana Rona • Ricardo Herdeiro • Cristiane Juliano Mathias •

Fernando Araripe Torres • Marcos Dias Pereira •

Elis Eleutherio

Received: 14 September 2014 / Accepted: 5 January 2015

� Springer Science+Business Media Dordrecht 2015

Abstract Studies using different organisms revealed

that reducing calorie intake, without malnutrition,

known as calorie restriction (CR), increases life span,

but its mechanism is still unkown. Using the yeast

Saccharomyces cerevisiae as eukaryotic model, we

observed that Cu, Zn-superoxide dismutase (Sod1p) is

required to increase longevity, as well as to confer

protection against lipid and protein oxidation under

CR. Old cells of sod1 strain also presented a premature

induction of apoptosis. However, when CTT1 (which

codes for cytosolic catalase) was overexpressed, sod1

and WT strains showed similar survival rates. Fur-

thermore, CTT1 overexpression decreased lipid per-

oxidation and delayed the induction of apoptotic

process. Superoxide is rapidly converted to hydrogen

peroxide by superoxide dismutase, but it also under-

goes spontaneous dismutation albeit at a slower rate.

However, the quantity of peroxide produced from

superoxide in this way is two-fold higher. Peroxide

degradation, catalyzed by catalase, is of vital

importance, because in the presence of a reducer

transition metal peroxide is reduced to the highly

reactive hydroxyl radical, which reacts indiscrimi-

nately with most cellular constituents. These findings

might explain why overexpression of catalase was

able to overcome the deficiency of Sod1p, increasing

life span in response to CR.

Keywords Calorie restriction � Ctt1 � Life span �Saccharomyces cerevisiae � Sod1p

Introduction

According to oxidative stress theory of aging, first

proposed by Harman in 1954, aging is correlated with

the accumulation of cellular damages triggered by

reactive oxygen species (ROS) produced by normal

cell metabolism. As the organism ages, antioxidants

decrease, increasing oxidative damage (Harman

2006). A large amount of data about the oxidative

cumulative values in healthy individuals related to age

has been shown (reviewed by del Valle 2011). In

general, the oxidative stress theory of aging was

confirmed, but some studies presented conflicting and

contradictory results concerning the level of antioxi-

dants in older volunteers (Mecocci et al. 2000; Ozbay

and Dulger 2002). Due to the complexity of higher

eukaryotic systems, it is appropriate to choose a

relatively simple, but still relevant, model system for a

G. Rona (&) � R. Herdeiro � C. J. Mathias �M. D. Pereira � E. Eleutherio

Department of Biochemistry, Institute of Chemistry,

Federal University of Rio de Janeiro, Avenida Athos da

Silveira Ramos 149, Bloco A, Lab 547, Rio de Janeiro,

RJ 21941-909, Brazil

e-mail: rona.germana@gmail.com

F. A. Torres

Department of Cellular Biology, Institute of Biology,

University of Brasılia, Brasılia, DF, Brazil

123

Biogerontology

DOI 10.1007/s10522-015-9550-7

deeper understanding of the role of oxidative stress in

longevity. Yeast can go through the aging process by

many ways, the most important are the chronological

(CLS) and replicative aging (RLS). Yeast CLS is a

measure of the survival of a non-dividing population

of cells (Parrella and Longo 2008). The RLS deter-

mines the number of divisions an individual mother

cell undergoes before death, the daughter cell, obtains

the undamaged material (Carmona-Gutierrez and

Buttner 2014).

In the past, studies using the yeast Saccharomyces

cerevisiae enabled major breakthroughs in the under-

standing of basic cellular and molecular processes

(Botstein and Fink 2011). Today, the use of yeast is

undergoing a ‘rebirth’ in both fundamental and

applied research. Studies of budding yeast have made

immense contributions to our understanding of the

aging process (Kaeberlein 2010). The reasons for this

success are experimental tractability, especially in

applying classical and molecular genetic methods to

associate genes with proteins and functions within the

cell.

Another reason for its applicability in aging-

related research is that some pathways involved in

degenerative diseases are conserved from yeast to

human. One-third of genes involved in human

diseases have S. cerevisiae orthologues. A common

strategy used to understand the molecular events of

diseases is the expression of human disease proteins

in yeast. S. cerevisiae also offers an attractive cellular

environment to investigate aging disease-related

proteins that have no apparent homologous counter-

part in yeast, holding great promises for the eluci-

dation of the molecular processes of these diseases

and the development of new therapies (Khurana and

Lindquist 2010; Mirisola et al. 2013). As the S.

cerevisiae is the most investigated eukaryotic micro-

organism, functional genomics techniques and bio-

informatics analysis of the transcriptome, proteome,

metabolome and interactome were originally devel-

oped using these yeast. This has resulted in dat-

abases, such as http://www.yeastgenome.org, that

represent valuable sources for research on aging.

Yeast cells are typically grown in media containing

high levels of glucose (2 %) and amino acids.

However, independent studies have shown that RLS

and CLS can be increased by reducing either glucose

or amino acid concentration (or both) (Mannarino

et al. 2008; Reverter-Branchat et al. 2007). In our

lab, we have used this microorganism to understand

the mechanism by which calorie restriction (CR)

increases life span (Mannarino et al. 2008; Huberts

et al. 2014). This intervention is able to extend the

life span of organisms ranging from yeast to mam-

mals and is thought to produce the higher increase in

human life expectancy (Baur 2010). However, the

potential significance of CR on human lifespan was

put in check in light of the recently published

research on CR in primates, which suggests that

reduction in calories intake does not affect life span

(Mattison et al. 2012). In 2009, another study con-

cluded that CR did extend life in rhesus monkeys

(Colman et al. 2009). Genetics probably explains part

of the variation between the monkey studies,

increasing the importance of the use of a less com-

plex model, such as yeast. Although the mechanism

by which CR increases life span is not completely

clear, it well known that oxidative damages are

reduced in calorie-restricted organisms (Agarwal

et al. 2005; Poljsak 2011). CR seems to induce

adaptive cell responses, which promote increased

resistance to adverse conditions, thus delaying the

onset of age-associated damages and extending life

span (Ribaric et al. 2012).

In this work, we analyzed if CTT1 overexpression,

which codes for a cytoplasmic catalase, would be able

to surpass Sod1p deficiency to increase life span of S.

cerevisiae in response to CR. This yeast, like most

other eukaryotes, possesses two isoforms of superox-

ide dismutase, the Cu/Zn-Sod1p and the Mn-Sod2p.

Both remove superoxide radical by converting it to O2

and H2O2, which must be further disproportionated by

catalases and peroxidases (Herrero et al. 2008).

Peroxide is very dangerous because in the presence

of some metals produces hydroxyl radical, one of the

most reactive and toxic free radical. While Sod2p

stays in mitochondria, Sod1p is found in cytoplasm,

nucleus and in the mitochondrial intermembrane

space, comprising the greatest proportion of the total

superoxide dismutase (Miao and St. Clair 2009).

Furthermore, Sod1p presents a pronounced physio-

logical significance and therapeutic potential, being

involved in diseases frequently associated with aging,

such as familial amyotrophic lateral sclerosis (FALS),

Parkinson’s disease and several neurological disorders

(Fukai and Ushio-Fukai 2011).

Biogerontology

123

Materials and methods

Yeast strains and plasmid construction

CTT1 was amplified from genomic DNA of wild-type

strain BY4741 (MAT a his3 leu2 met15 ura3—

Euroscarf, Germany) by PCR, using Platinum Taq

DNA Polymerase High Fidelity (Invitrogen) and

primers CTT1-F (50-ggatccAAATGAACGTGTTCG

GTAAAAAAG) and CTT1-R (50-ggatccTTAATTGG-

CACTTGCAATGGAC), including a BamHI site

(lower case letters). The PCR product was purified

using the illustra GFX PCR DNA and Gel Band

Purification kit (GE Healthcare) and cloned into

pGEM-T vector (Promega). After digestion with

BamHI, a 1.7 Kb purified amplicon was inserted into

the YEp351-PGK (de Moraes et al. 1995), previously

digested with BglII, to obtain pPGK-CTT1. Plasmids

were propagated in Escherichia coli XL10 Gold grown

on LB medium with 100 lg/ml ampicillin. Standard

molecular biology techniques were used (Sambrook

et al. 1989). Following, pPGK-CTT1, harboring CTT1

under control of the PGK1 promoter and LEU2 as

selectable marker, was used to transform both BY4741

and its isogenic sod1 strain (sod1::KanMX4—Euro-

scarf, Germany), using the lithium acetate protocol

(Gietz et al. 1992).In the same way BY4741 and its

isogenic sod1 were also transformed with the YEp351-

PGK vector to use as a control. Transformants were

selected in synthetic medium (0.67 % yeast nitrogen

base without aminoacids, 0.01 % histidine, 0.01 %

uracil, 0.01 % methionine and 2 % agar) supple-

mented with 2 % glucose (SD 2 %).

Growth conditions, life span and oxidative damage

For the experiments, cells were grown in liquid media

SD 2 % or SD 0.5 % (containing 0.5 % glucose instead

of 2 %), until the middle of exponential growth phase

(OD570 = 0.6), with the ratio of flask volume/medium

of 5/1, at 28 �C and 160 rpm. To analyze life span and

oxidative damage accumulation as a function of time,

cells were switched from culture media to water (non-

proliferating conditions) and incubated at 37 �C/

160 rpm (Mannarino et al. 2008, 2011). Cell viability

was determined over time by standard dilution plate

counts on solid YPD medium (2 % glucose, 1 % yeast

extract 2 % peptone). Viability was expressed as the

percentage of colony forming units. The oxidant-

sensitive probe 2,7-dichlorofluorescin diacetate

(DCFDA) was used to measure the level of oxidative

stress (Mannarino et al. 2008). Protein carbonylation

and lipid peroxidation were determined as previously

described (Adamis et al. 2007). Slot blot using an anti-

DNP antibody revealed ROS-dependent protein car-

bonylation. Lipid oxidation was measured by TBARS

(thiobarbituric acid reactive species) method, which

detects malondialdehyde, a final product of lipid

peroxidation. Cells were centrifuged and washed with

cold distilled water. The pellets were resuspended in

0.5 ml of 10 % TCA (w/v) followed by addition of

1.5 g of glass beads. The samples were lysed by 6

cycles of 20 s agitation on a vortex followed by 20 s on

ice. Extracts were centrifuged and the supernatant

mixed with 0.1 ml of 0.1 M EDTA and 0.6 ml of 1 %

(w/v) thiobarbituric acid prepared in 0.05 M NaOH.

The reaction mixture was incubated in a boiling water

bath for 15 min and, after cooling, the absorbance was

measured at 532 nm (Steels et al. 1994).

Enzyme activities and apoptotic markers

Extracts for enzymatic determinations were obtained by

the disruption of cells with glass beads in 0.1 M Tris–

HCl buffer, pH 6.0 (Pereira et al. 2003). Protein

concentration was determined according to (Stickland

1951), using bovine serum albumin as standard. The

catalase activity was determined based on H2O2

consumption (Aebi 1964). Apoptosis was analyzed by

caspase activity and Annexin V-FITC staining. Caspase

assays were based on the measurement of the hydrolysis

of the peptide substrate acetyl-Asp-Glu-Val-Asp

p-nitroanilide, resulting in the release of the p-nitroan-

iline (Caspase assay kit, CASP-3-C, Sigma-Aldrich,

USA). Annexin V conjugated with FITC fluorochrome

staining (CLONTECH Laboratories, Inc.) was per-

formed as previously described (Gomes et al. 2008). To

determine frequencies of phenotypes, at least 300 cells

of three independent experiments were evaluated.

Results and discussion

Irrespective of the model organism, C. elegans, Dro-

sophila or yeast, the reduction of oxidative damage is

one of the mechanisms that extend life span in response

to CR (Shimokawa et al. 2008). Oxygen consumption

and intracellular levels of ROS are higher in calorie-

Biogerontology

123

restricted than in non-restricted yeasts, since there is a

higher respiratory chain activity under CR (Agarwal

et al. 2005; Mesquita et al. 2010). Because of such mild

stress, some antioxidant systems are overexpressed

(Agarwal et al. 2005), making calorie-restricted yeasts

more efficient in the destruction of ROS.

In this work, to understand the biological mecha-

nism underlying the anti-aging action of CR, the

relationship between antioxidants systems and some

markers of oxidative stress in calorie-restricted yeasts

was investigated. For this, we used a mutant yeast

strain unable to express SOD1, containing or not the

high copy plasmid Yep351PGK-CTT1, which endows

cells with constitutive CTT1 overexpression. The

catalase overexpression was confirmed in the mutants

WT CTT1 and sod1 CTT1, under caloric restriction,

showing that both lineages presented similar catalase

activities (Table 1). These scavenging enzymes were

chosen because it was previously found that their

expressions are induced in CR (Agarwal et al. 2005).

Yeasts grew in media containing 2 or 0.5 % glucose

(to simulate CR) until the middle of exponential

growth phase and shifted to water for measuring how

long a cell can survive in a non-dividing state. This

strategy has been applied by us and other groups

(Fabrizio and Longo 2003; Harris et al. 2005;

Mannarino et al. 2008, 2011; Piper 2006). In the wild,

an environment that lacks nutrients is common for

microorganisms and even to certain mammals (hiber-

nation of bears and rodents). When yeast cells are

incubated in water, they are not starving, because they

metabolize the intracellular stored nutrients.

As expected, longevity of calorie-restricted cells was

significantly higher than non-restricted cells (Fig. 1). At

high levels of glucose (2 %), yeasts use glucose to obtain

energy exclusively via glycolysis, their cellular defense

systems are repressed and, for this reason, they are very

sensitive to a severe oxidative stress, as occurs during

incubation in water at 37 �C. Damages accumulate over

time in water because cell division and new synthesis are

not occurring. Furthermore, when cells are shifted to

37 �C, the accumulation of oxidative damages and

consequently the aging process are accelerated. Accord-

ing to previous results, differences in life span measured

at 28 �C are reproducible at 37 �C, i.e., the differences in

life span between a mutant and its parental strain are the

same at both temperatures but appear earlier at 37 �C

(Piper 2006; Mannarino et al. 2008, 2011). As can be seen

in Fig. 1a, both WT and sod1 mutant strains presented a

similar life span when incubated in water at 37 �C when

previously grown in 2 % glucose. This result had already

been observed when cells were grown and kept in rich

(YPD) media containing 2 % glucose at 28 �C for several

days: cells deficient in Sod1p showed a life span similar to

WT (Demir and Koc 2010). However, CR was not able to

increase cell longevity in the absence of Sod1p (Fig. 1b).

Next, it was investigated the level of intracellular

oxidation as well as the accumulation of oxidized

Fig. 1 Sod1p is required for CR-mediated CLS extension. Life

span of non-CR (a) or CR cells (b). Cells were grown in SD 2 %

(a) or SD 0.5 % medium (b), until the middle of exponential

growth phase and washed with water. For life span measure-

ments, these cultures were then transferred to water and

incubated at 37 �C/160 rpm. Cellular viability was measured

by standard dilution plate counts and expressed as the

percentage of the colony-forming units at time 0 h of incubation

in water. Values are mean ± SD of three independent

experiments

Table 1 Catalase activity

Strains U/mg ptn

WT (BY4741) 1.3 ± 0.1

sod1 1.5

WT CTT1 38.4 ± 0.5

sod1 CTT1 24.1 ± 1.0

Catalase activity was determined on the basis of H2O2

consumption as described in ‘‘Materials and methods’’

section. The results represent the mean ± standard deviation

of at least three independent experiments

Biogerontology

123

proteins and lipids during incubation of cells in water

(Fig. 2). Higher levels of protein oxidation or lipid

peroxidation accelerate the progress of aging and

neurodegenerative diseases (Radak et al. 2011). To

quantify the production of ROS, we used the DCFDA

assay. Intracellular fluorescence due to dichlorofluores-

cin oxidation has been generally considered as a

representation of cellular ROS concentration (Bar

2002), especially for measuring hydrogen peroxide

concentration (Carter et al. 1994). As can be seen in

Fig. 2a, ROS accumulation was higher in the mutant,

being more evident after 72 h in water. The accumula-

tion of ROS caused a high increase in the levels of protein

carbonyl groups in both strains but in sod1 mutant the

increase was much more impressive (Fig. 2b). Sod1p

deficiency also led to a higher increase in lipid oxidation

(Fig. 2c). Among the parameters used to analyze the

oxidative damage, the level of carbonylation showed the

highest increase in response to aging. It is known that

cells degrade oxidized proteins within hours and days,

whereas lipid peroxidation products are detoxified

within minutes (Dalle-Donnea et al. 2003). Thus, in

accordance with Agarwal et al. (2005) that correlated the

increased life span of calorie-restricted yeasts with the

induction of expression of scavengers, including SOD1,

by CR, our results demonstrated that calorie-restricted

cells of sod1 mutant strain died much sooner than WT,

which seems to be related to a higher accumulation of

oxidative damages to lipids and proteins.

The majority of ROS production occurs in the

electron transport chain of the mitochondria (Sheu

et al. 2006). In this process, one molecule of oxygen

receives four electrons and then can be reduced to

water, but some of the electrons escape to form ROS. In

higher eukaryotes, superoxide is thought to be gener-

ated when electrons are transferred to and from

ubiquinone, specifically at complex I and complex

III. S. cerevisiae lacks complex I, but has NADH

dehydrogenases located at the mitochondrial inner

membrane space able to catalyze the partial reduction

of oxygen to superoxide in this compartment (Herrero

et al. 2008). To maintain redox homeostasis, multiple

enzyme defense system, like superoxide dismutases

and catalases enzymes, is present in the cells. By the

action of Sod, superoxide, which stays in the compart-

ment in which it was generated, is rapidly converted to

oxygen and hydrogen peroxide. According to litera-

Fig. 2 Oxidative stress in calorie-restricted cells, measured as

fold increase in fluorescence (intracellular oxidation) (a),

protein carbonylation (b) or lipid peroxidation (c). Cells were

grown in SD 0.5 % medium, harvested in mid-log growth phase

and then stressed by incubating them in water at 37 �C/160 rpm.

Oxidative damages were analyzed over time. The results were

expressed as a ratio between fluorescence, lipid peroxidation or

protein carbonylation of stressed cells and control situation

(time zero of incubation in water) of each strain. The results

represent the mean ± SD of at least three independent

experiments. *p \ 0.05 (sod1 vs. WT)

Biogerontology

123

ture, superoxide dismutase is a major antioxidant in

yeast stationary phase, playing a very important role in

its survival (Longo et al. 1996). Stationary phase is

characterized by nutrient limitation, no cell division

and mitochondrial respiration. The fast loss of viability

of cells lacking Sod1p observed under normal but not

low aeration levels suggests that Sod2p is able to

prevent migration of ROS from the mitochondria to the

cytoplasm only under lower oxygen levels (Longo

et al. 1996). Peroxide produced by superoxide

dismutases must be fully reduced to water through

catalase or peroxidases to avoid production of hydro-

xyl radical, the most reactive and dangerous radicals.

Superoxide also undergoes spontaneous dismutation

albeit at a slower rate. However, the quantity of

peroxide produced from superoxide in this way is two-

fold higher (Fernandes et al. 2007). However, the

differences between the rates of conversion of the

enzymatic and non-enzymatic reactions, high rates of

peroxide production could be achieved from high

concentrations of superoxide (as under deficiency of

Sod1p) in proper intracellular conditions (pH, metal

concentration). Furthermore, the high sensitivity of the

sod1 mutant to oxidative stress is now largely attrib-

uted to its inability to assemble iron-sulfur proteins,

leading to high intracellular level of free iron, trigger-

ing to a higher possibility of Fenton reactions occur

(Srinivasan et al. 2000). Thus, next, we investigated if

catalase overexpression, one of the enzymes respon-

sible for the conversion of peroxide to water and

oxygen, might overcome Sod1p deficiency, allowing

sod1 mutants enjoy the benefits of CR. Yeast cells

overexpressing cytosolic catalase (WT CTT1 and sod1

CTT1) were obtained by introducing pPGK-CTT1 into

WT and sod1 strains. To test CTT1 overexpression, all

strains were grown in SD 0.5 % medium until mid-log

phase, a growth condition that increases life span in

water. As expected, catalase activity was very low and

practically the same in both WT and sod1 strains

(specific activity around 1 U/mg protein). However, in

sod1 CTT1 and WT CTT1 strains, catalase activities

were 20 to 30-fold higher than sod1 and WT stains,

respectively, confirming that cells possessing pPGK-

CTT1 overexpress CTT1 (Table 1).

b Fig. 3 Effect of CTT1 overexpression on life span (a), protein

carbonylation (b) or lipid peroxidation (c). WT CTT1 and sod1

CTT1 cells were grown in SD 0.5 % medium up to mid-log

growth phase, washed and stressed by shifting them to water at

37 �C/160 rpm. Cellular viability was measured by standard

dilution plate counts and expressed as the percentage of the

colony-forming units at time 0 h of incubation in water.

Oxidative damages were analyzed over time. The results were

expressed as protein carbonylation and lipid peroxidation of

stressed cells and control situation (time zero of incubation in

water) of each strain. Protein carbonylation was detected by a

slot blot using an anti-DNP antibody. The levels of lipid

peroxidation were determined through TBARS method and the

results were expressed as a ratio between the level of lipid

peroxidation of stressed cells and control situation (time zero of

stress—incubation in water) of each strain. Values are

mean ± SD of three independent experiments

Biogerontology

123

According to Fig. 3a, when catalase was overex-

pressed, calorie-restricted cells of sod1 and WT strains

showed similar survival rates and levels of oxidative

damage during incubation in water. The life span

showed by sod1 CTT1 strain was higher than its parental

sod1 (Fig. 1b). However, overexpression of CTT1 only

improve longevity of calorie-restricted WT cells after

48 h of incubation in water. After 24 h, 35 % of WT

CTT1 cells were still alive (Fig. 3a) whereas its parental

strain WT showed a survival rate twofold higher

(Fig. 1b). Recently, several findings indicate that ROS

may serve as essential signaling molecules that promote

metabolic health and longevity (Ristow and Schmeisser

2011). Conditions involved with ROS generation, such

as heat and oxidative mild-stresses, lead to Hsf activa-

tion (Liu and Thiele 1996) and Yap1 nuclear localiza-

tion (Rodrigues-Pousada et al. 2010). It is well known

that increased ROS levels are detrimental. However,

lowering ROS levels below the homeostatic set point

may interrupt the physiological role of oxidants in

cellular processes such as induction of defense systems

against stress. Consistently, an excess of antioxidant, as

possible occurred with WT CTT1 at the beginning of

incubation in water, might prevent these ROS signals

and interfere with the life-span-extending capabilities of

CR. Only after 48 h in a non-dividing condition, when

high levels of oxidative damage had accumulated

(Fig. 2), CTT1 overexpression was suitable to counter-

act and regulate overall ROS levels to maintain

physiological homeostasis. Corroborating the idea of

the role of ROS as second messengers involved in signal

transduction, it was recently shown that deficiency in

peroxisomal catalase extended chronological lifespan of

yeast by inducing elevated levels of hydrogen peroxide,

which, in turn, activated superoxide dismutases (Mesq-

uita et al. 2010). As can be seen in Fig. 3b and C,

overexpressing CTT1 sod1 strain pre-grown under CR

also showed lower increases in levels of protein

carbonyl groups and lipid peroxidation during incuba-

tion in water than its parental strain sod1 (Fig. 2b, c).

These result leads us to conclude that overexpression of

catalase is able to overcome the deficiency of Sod1p in

the mechanism in which CR prolongs life span.

Saccharomyces cerevisiae shows features of apop-

totic death during chronological aging, as a form of

removal of damaged cells (Herker et al. 2004). To

investigate whether apoptosis might contribute to the

lower longevity shown by sod1 cells, we analyzed two

apoptotic markers, caspase activity and externalization

of phosphatidylserine. In yeast, phosphatidylserine

externalization can be detected by FITC labeled annexin

V staining (Gomes et al. 2008). Concomitantly, cells

must be checked for membrane integrity by incubation

with propidium iodide. Caspases (Cysteine-requiring

Aspartate protease) are a family of proteases that

mediate cell death and are important to process of

apoptosis. Homologues of metazoan caspases have been

identified on the yeast genome and shown to be

implicated in hydrogen peroxide induced apoptosis

(Fabrizio and Longo 2008). Caspase 3 (also referred to

Fig. 4 Markers of apoptosis. Cells were grown in SD 0.5 %

medium until mid-log phase, washed and stressed by incubating

them in water at 37 �C/160 rpm up to 24 h. Caspase activity was

determined as described in Methods and expressed as a ratio

between activity of stressed cells and control situation (time zero

of stress—incubation in water) of each strain. To determine

percentage of cells showing externalization of phosphatidylser-

ine, at least 300 cells of three independent experiments were

evaluated. Phenotype (%) = [(number of cells with apoptotic

markers)/(number of total cells)] 9 100. Values are

mean ± SD of three independent experiments

Biogerontology

123

as CPP32, Yama and apopain) is a member of the CED-

3 subfamily of caspases and plays a central role in

mediating nuclear apoptosis including chromatin con-

densation and DNA fragmentation. According to our

results, apoptosis was induced earlier during incubation

in water in cells lacking Sod1p (Fig. 4). Caspase activity

was not induced in WT but increased 50 % in sod1

yeasts. In addition, more than 20 % of sod1 cells were

stained with annexin V, what was not observed in WT.

However, CTT1 overexpression abolishes the premature

induction of apoptosis observed in sod1 strain, which

might explain the increased longevity of sod1 CTT1.

When sod1 cells were engineered to overexpress CTT1,

oxidative damage decreased, delaying the induction of

apoptosis and the onset of morbidity.

Conclusion

Our results confirmed that CR augments ROS defense

in S. cerevisiae, used as a model in this work, by a

Sod1p dependent mechanism. On the other hand, the

overexpression of CTT1 was able to overcome the

absence of Sod1p. In fact, the increase of life span

occurred because this overexpression might play an

important role in the cell membrane protection,

decreasing the oxidative damages and delaying the

induction of apoptotic process. On the order hand, it is

important to observe that the excess of catalase can be

harmful for the cell, getting the cell to an unbalanced

scenario between antioxidant defense system and

ROS, deregulating the physiological homeostasis.

Acknowledgments This work was supported by grants from

CAPES and CNPq.

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