Báo cáo khoa học: Yeast oxidative stress response Influences of cytosolic thioredoxin peroxidase I and of the mitochondrial functional state pot

Our results indicated two possibilities not mutually exclusive for this cTPxI-dependent protection: a the dual functions of cTPxI as peroxidase and as molecular chaperone, suggested by m

Influences of cytosolic thioredoxin peroxidase I and of the

mitochondrial functional state

Ana P D Demasi1, Gonc¸alo A G Pereira1and Luis E S Netto2

1 Departamento de Gene´tica e Evoluc¸a˜o – IB – UNICAMP, Campinas, Brazil

2 Departamento de Gene´tica e Biologia Evolutiva – IB – USP, Sa´o Paulo, Brazil

Aerobic organisms are constantly exposed to reactive

oxygen species (ROS), generated as normal metabolism

byproducts, especially by respiration [1,2] To

modu-late ROS concentrations and to counteract their

dele-terious effects, there are several antioxidant systems

with an apparent functional redundancy, which may

provide an evolutionary advantage Saccharomyces cerevisiae cells possess multiple H2O2 detoxifying enzymes, such as catalases, cytochrome c peroxidase, glutathione peroxidases, glutaredoxins and peroxire-doxins, described as many isoforms and in distinct cel-lular compartments [3–6] So far, their specific roles

Keywords

hydrogen peroxide; gene expression;

mitochondrial dysfunction; oxidative stress;

thioredoxin peroxidase

Correspondence

G Amarante Guimara˜es Pereira, Laborato´rio

de Genoˆmica e Expressa˜o – IB UNICAMP,

CP 6109, CEP 13083–970, Campinas-SP,

Brazil

Fax: + 55 19 37886235

Tel: + 55 19 37886237 ⁄ 6238

E-mail: goncalo@unicamp.br

(Received 30 September 2005, revised

12 December 2005, accepted 20 December

2005)

doi:10.1111/j.1742-4658.2006.05116.x

We investigated the changes in the oxidative stress response of yeast cells suffering mitochondrial dysfunction that could impair their viability First, we demonstrated that cells with this dysfunction rely exclusively on cytosolic thioredoxin peroxidase I (cTPxI) and its reductant sulfiredoxin, among other antioxidant enzymes tested, to protect them against H2O2 -induced death This cTPxI-dependent protection could be related to its dual functions, as peroxidase and as molecular chaperone, suggested by mixtures of low and high molecular weight oligomeric structures of cTPxI observed in cells challenged with H2O2 We found that cTPxI deficiency leads to increased basal sulfhydryl levels and transcriptional activation of most of the H2O2-responsive genes, interpreted as an attempt by the cells

to improve their antioxidant defense On the other hand, mitochondrial dysfunction, specifically the electron transport blockage, provoked a huge depletion of sulfhydryl groups after H2O2treatment and reduced the H2O2 -mediated activation of some genes otherwise observed, impairing cell def-ense and viability The transcription factors Yap1 and Skn7 are crucial for the antioxidant response of cells under inhibited electron flow condition and probably act in the same pathway of cTPxI to protect cells affected by this disorder Yap1 cellular distribution was not affected by cTpxI defici-ency and by mitochondrial dysfunction, in spite of the observed expression alterations of several Yap1-target genes, indicating alternative mechanisms

of Yap1 activation⁄ deactivation Therefore, we propose that cTPxI is specifically important in the protection of yeast with mitochondrial dys-function due to its dys-functional versatility as an antioxidant, chaperone and modulator of gene expression

Abbreviations

AhpC, alkyl hydroperoxide reductase; cTPxI, cytosolic thioredoxin peroxidase I, which is synonymous with Tsa1 and YML028W; 2-Cys Prx, peroxiredoxins with two conserved cysteines involved in the catalytic mechanism; DTNB, 5,5¢-dithio-bis(2-nitrobenzoic acid); FCCP,

p-trifluoromethoxycarbonylcyanide phenylhydrazone; NP-SH, nonprotein sulfhydryl groups; PB-SH, protein bound sulfydryl group; PKA, protein kinase A; Prxs, peroxiredoxins; ROS, reactive oxygen species; t-BOOH, t-butylhydroperoxide; TSA, thiol-specific antioxidant; Ybp1, Yap1-binding protein.

have not been well established and are suggested to be

related to their differential regulation

The transcription factors Yap1, Skn7, Msn2 and

Msn4 are the main regulators of S cerevisiae oxidative

stress response [7–10] Yap1 confers to cells the ability

to tolerate oxidants like H2O2, t-butyl hydroperoxide,

diamide, diethylmaleate and cadmium [11] by the

activa-tion of the expression of genes encoding most

anti-oxidant enzymes and components of the cellular

thiol-reducing pathways, comprising approximately 32

proteins of the H2O2stimulon [12] Skn7 co-operates in

the activation of at least 15 of the Yap1 target proteins

in response to H2O2and t-butyl hydroperoxide, but not

to cadmium [7] Contrary to Yap1, this transcriptional

regulator does not participate in the regulation of

meta-bolic pathways that regenerate the main cellular

redu-cing power, glutathione and NADPH [7] Msn2 and

Msn4 activate genes whose promoters contain the stress

responsive element (STRE: CCCCT) after several

envi-ronmental challenges, including oxidative stress Despite

an overlap with the Yap1 regulon (eight proteins), the

Msn2⁄ 4 regulon comprises a small number of

antioxid-ant enzymes, but several heat-shock proteins,

meta-bolism enzymes and proteases, and is involved with

activities of the ubiquitin and proteasome degradation

pathways [13] Msn2⁄ 4 are negatively regulated by the

Ras-cAMP-protein kinase A (PKA) pathway [14], which

has been suggested to negatively influence also

Yap1-regulated transcription [8,15]

The involvement of mitochondria in the response of

yeast to oxidative stress is not well understood, despite

the fact that these organelles are the primary cellular

source of ROS In S cerevisiae cells, external

mito-chondrial NADH dehydrogenases [16], coenzyme Q

[17] and succinate dehydrogenase [18] were identified

as sites of ROS production in mitochondria

Muta-tions or drugs that block terminal steps of the

respirat-ory chain further increase ROS generation due to the

accumulation of reduced electron carriers [19–21]

Even so, it was demonstrated that mitochondrial

func-tion is required for resistance to oxidative stress

[22,23]

Peroxiredoxins (Prxs) are abundant, ubiquitously

distributed peroxidases that reduce H2O2 and

peroxy-nitrite at the expense of thiol compounds [24–26] They

can be divided into 1-Cys and 2-Cys Prxs, based on

the number of cysteine residues involved in catalysis It

has been shown that 2-Cys Prxs participate in the

regulation of H2O2-mediated signal transduction [27–

32] In addition, two recent reports have demonstrated

that 2-Cys Prxs can act alternatively as peroxidases

and as molecular chaperones [33,34] The peroxidase–

chaperone functional switch is established by a shift of

the cTPxI oligomeric state from low to high molecular weight complexes, which is triggered by oxidative and heat stress [33,34]

Cytosolic thioredoxin peroxidase I (cTPxI), encoded

by TSA1, is a 2-Cys Prx and is one of the five Prxs described in S cerevisiae It was shown that cTPxI is essential for the H2O2 defense of yeast with dysfunc-tional mitochondria [35] Here, we describe results indicating that cells with this dysfunction rely exclu-sively on cTPxI and its reductant sulfiredoxin, among other antioxidant enzymes tested, to protect them against H2O2-induced death Our results indicated two possibilities (not mutually exclusive) for this cTPxI-dependent protection: (a) the dual functions of cTPxI

as peroxidase and as molecular chaperone, suggested

by mixtures of low and high molecular weight oligo-meric structures observed in cells challenged with

H2O2 and (b) the capacity of cTPxI to interfere with the expression of various Yap1-target genes

Results Effects of gene deletion and mitochondrial perturbation on the oxidative stress response

We have previously shown that cTPxI is an important component of the defense of cells with mitochondrial dysfunction against H2O2 [35] Using the same experi-mental approach, we have compared the sensitivities of tsa1D and other mutants deficient in different antioxid-ant enzymes to H2O2 when mitochondrial function was affected by antimycin A treatment (inhibitor of complex III) We observed that loss of any of these enzymes, cytosolic thioredoxin peroxidase II, alkyl hydroperoxide reductase, mitochondrial thioredoxin peroxidase I, cytochrome c peroxidase, glutathione reductase, catalase T and catalase A, did not alter the growth, either after the single treatments or after the associations of both treatments (Fig 1) On the other hand, the deficiency of sulfiredoxin, a low molecular weight protein that reduces the overoxidized forms of cTPxI [36], did alter the growth when cells were trea-ted with antimycin A plus H2O2 (Fig 1) Therefore, the response of yeast with dysfunctional mitochondria was very dependent on cTPxI and its reductant sulfi-redoxin, among other antioxidants

Next, we evaluated the behavior of tsa1D cells and other mutants in response to H2O2 when respiration was altered with p-trifluoromethoxycarbonylcyanide phenylhydrazone (FCCP), an oxidative phosphoryla-tion uncoupler that can carry protons across the mito-chondrial inner membrane, thus promoting proton gradient collapse Contrary to antimycin A, FCCP

treatment accelerates the electron flow through the

respiratory chain and diminishes endogenous ROS

generation by mitochondria [37–39] No increased

sen-sitivity to H2O2 could be detected in any of the strains

treated with FCCP (Fig 1)

The phenotype of mutant strains in response to

other oxidants besides H2O2 was also investigated All

strains presented similar sensitivity to

t-butylhydro-peroxide (t-BOOH) treatment, but tsa1D was slightly

more sensitive than the others to this oxidant when

antimycin A was also present in the media (Fig 1)

Interestingly, tsa1D cells were not sensitive to diamide,

even in the presence of antimycin A (Fig 1) Only

glr1D was more sensitive than wild-type cells to

di-amide and this effect was increased when cells were

also exposed to antimycin A (Fig 1) This result was

expected, as diamide only induces generation of

disul-fide bridges [40] and glutathione reductase reduces the

disulfide form of glutathione This point will be further

explored in the discussion section

In summary, the results presented here indicated

that cTPxI exhibits a very specific defense of yeast with

dysfunctional mitochondrial in situations in which this

organelle presents electron transport impediment and⁄

or produces high levels of superoxide

Oxidation of sulfhydryl groups

Sulfhydryl groups, including nonprotein (NP-SH),

mostly represented by glutathione, and protein bound

(PB-SH), are abundant in cells and can be oxidized by

ROS Therefore, they have been widely used as

indica-tors of oxidative stress [41–43] To determine whether

TSA1 deletion and the mitochondrial dysfunction can

generate stressful conditions, the levels of sulfhydryl groups in the reduced state were evaluated

The first remarkable observation was that tsa1 mutant presented a pronounced increase in basal sul-fhydryl groups compared with the wild-type strain, especially in the NP-SH content (Fig 2) In spite of the high basal sulfhydryl content present in tsa1D cells, exposure to H2O2 promoted a significant loss of these

WT1

srx1

tsa1

tsa2

ahp1

prx1

ccp1

glr1

WT1

WT2

ctt1

cta1

l o t n

o H 2 O 2 a n t i A H 2 O 2 +

A i t n a

H 2 O 2 + P C F P C

F t- B O H t- B O O H

A i t n a + d i a m i d e

e d i m a i d A i t n a +

∆

∆

∆

∆

∆

∆

∆

∆

∆

Fig 1 Comparison of yeast mutants’ sensitivities to several stressful conditions Growth of the strains BY4741, wild-type 1 (WT1), YPH250, wild-type 2 (WT2) and mutants indicated on YPD plates containing no chemicals as a control, 1.2 m M H2O2, 1.2 m M t-BOOH, 1.2 m M diamide, 0.1 lgÆmL)1antimycin A (anti A), 2.5 lgÆmL)1FCCP, 5.0 lgÆmL)1singly or in association For each strain, 12 lL of overnight culture diluted to 0.2 OD 600nm units and four subsequent 1 : 5 dilutions were spotted on plates Growth was monitored after 2 days for all plates except for diamide, after 5 days Only the three last dilutions are represented in the figure.

B P B P B P B P B P B P 0 20 40 60 80 100 120 140 160

180

T W

tsa1

H l o r t n

c 2 O 2 A n t i A A n t i A

H + 2 O 2 F C P

P C F H + 2 O 2

∆

Fig 2 Comparison of sulfhydryl group levels in wild-type (WT) and tsa1D cells exposed to several stressful conditions Cell protein extracts of strains BY4741 (WT) and tsa1D, obtained after exposi-tion to 1.2 m M H2O2, 0.1 lgÆmL)1 antimycin A (anti A) or 2.5 lgÆmL)1FCCP, singly or in association, were assayed for thiol groups by spectrophotometric test using DTNB Absorbance was read at 412 nm Results are relative to the concentration of these groups in control cells (100%) that were not exposed to any agent, and represent average of 3 independent experiments PB, protein bound sulfydryl; NP, nonprotein sulfydryl.

groups, reaching levels similar to those observed in

wild-type cells, much less affected Antimycin A

treat-ment alone caused little increase of sulfhydryl groups

in both strains, when compared with the control

situ-ation However, the association of antimycin A with

H2O2 led to a huge depletion in NP-SH, as well as

PB-SH levels, only in cells lacking cTPxI (Fig 2)

Only a limited loss of sulfhydryl groups was observed

for both strains treated with FCCP alone, and no

additional decreases in these levels were found with

the addition of H2O2, even in cells lacking cTPxI

(Fig 2) These results indicated that tsa1 mutant cells

with dysfunctional mitochondria suffered intensive

oxidative stress only when this dysfunction is

accom-panied with electron flow obstruction and⁄ or

increased endogenous ROS production (antimycin A

treatment)

Switching of cTPxI oligomeric states in vivo

cTPxI and cTPxII can act as peroxidases and as

molecular chaperones, depending on changes of their

quaternary structures triggered by oxidative stress and

heat shock exposure [33,34] When cTPxI appears

mainly as oligomeric protein structures of low

molecu-lar weight, this protein possesses mainly peroxidase

activity, whereas high molecular weight complexes

behave mainly as chaperones [33,34] The specificity of

cTPxI in the protection of cells with dysfunctional

mitochondria (Fig 1) might be related to the ability of

this protein to possess these two activities Therefore,

we compared cTPxI oligomeric structures in vivo under

situations of normal and inhibited mitochondrial

func-tion, as it is hard to measure chaperone activity

in vivo

Under control conditions, cTPxI appeared as a

mix-ture of complexes with molecular weight below

272 kDa and after treatment of yeast cells with H2O2,

a considerable part of these species were converted to

HMW complexes of about 500 kDa or even higher

(Fig 3), as previously described [33,34] These switches

of cTPxI quaternary structures induced by H2O2 were

not affected by any of the inhibitors of mitochondrial

function (Fig 3) Similar results were obtained with

0.5 mm H2O2 alone or in association with the

mito-chondrial function inhibitors (not shown)

Since it was well demonstrated that the conversion of

cTPxI to different oligomerization states is implicated

with its chaperone⁄ peroxidase switching [33,34], we

suggest that the chaperone activity of this protein,

in addition to its peroxidatic function, is probably

involved with its specific role in the antioxidant defense

of yeast with mitochondrial dysfunction

cTPxI influences the expression of genes involved

in yeast oxidative stress response: mitochondrial function contribution

cTPxI participates of H2O2-mediated signaling proces-ses, including regulation of gene expression [27–30] Therefore, we have evaluated possible influences of cTPxI and of the functional state of the mitochondria in the expression of selected yeast antioxidant genes In this manner, we expected to obtain some clues to better understand cTPxI importance in the response of cells with mitochondrial dysfunction to oxidative stress

It could be readily observed that the expression levels

of several genes were increased in cells lacking cTPxI (Fig 4) Four gene subsets could be delineated: (a) genes with increased basal expression levels in tsa1D cells: GSH1, GSH2, GLR1, PRX1, SOD1, GPX2, AHP1, TRR1, SSA1; (b) genes with increased H2O2 -induced expression levels in tsa1D cells: CCP1, CTT1, TRX2, TRX3, SOD2, GRX5, POS5; (c) genes without expression alteration in tsa1D cells: ZWF1, IDP1, GPX3, HSP104 (not shown); and (d) genes without

545 272

132

66

45

(kDa)

controlH 2 O 2

Anti AAnti A + H

2

O 2

FCCP FCCP + H

2

O 2

Fig 3 Protein structures of cTPxI in vivo Native-PAGE analysis of crude protein extracts obtained from BY4741 (WT) cultures after exposing cells during 40 min to no agent as a control, 1.2 m M

H2O2, 0.1 lgÆmL)1 antimycin A (anti A), 1.2 m M H2O2 plus 0.1 lgÆmL)1antimycin A, 2.5 lgÆmL)1FCCP and 2.5 lgÆmL)1FCCP plus 1.2 m M H2O2, were separated on 9% native-PAGE and subjec-ted to western blotting with a polyclonal anti-cTPxI IgG.

basal

expression

levels in

tsa1∆ cells

Increased

H2O2

-induced

expression

levels in

tsa1∆ cells

Mitochondrial function

γ-glutamyl

Mitochondrial

Yap1

+/-Cytochrome c peroxidase

Msn2/4 +

Thioredoxin III

Manganese superoxide

+/-Glutaredoxin 5

Cytosolic thioredoxin

-Glutathione

-Coper/zinc superoxide

-Alkyl hydroperoxide

-1 2 3 4 5 6 7 8

Thioredoxin

+/-POS5

Mitochondrial

+/-Glutathione

-SSA1

Heat shock protein

Fig 4 Expression of genes in wild-type and tsa1D cells exposed to several stressful conditions Northern blot analysis of RNA isolated by the hot acid phenol method from yeast strains BY4741 (WT) and tsa1D grown on YPD to mid-log phase, treated during 40 min with no agent

as a control or with 1.2 m M H2O2and 0.1 lgÆmL)1antimycin A (anti A), singly or in association, as indicated in the figure The symbols –, + or ± in the last column denote absence, strong or mild influence of mitochondrial function on gene expression (comparison of band intensities between lanes 6 and 8).

detectable expression in both wild-type and tsa1D cells:

TSA2, CTA1, DOT5, TRX1, TTR1 (not shown) Genes

that belong to subsets (c) and (d) encode

glucose-6-phosphate dehydrogenase, mitochondrial isocitrate

de-hydrogenase, glutathione peroxidase III, heat shock

protein 104, thioredoxin peroxidase II, catalase A,

nuc-lear thioredoxin peroxidase, thioredoxin I and

glutare-doxin II, respectively Among genes described in subset

(a), GSH1, GPX2, AHP1 and TRR1 expression levels

were further induced by H2O2 (Fig 4) In addition,

most if not all genes that presented altered expression in

tsa1 mutant are regulated by Yap1 (Fig 4), indicating

that cTPxI could affect Yap1 activity Furthermore,

Skn7 co-operates in the control of many of these genes

(Fig 4) and constitute another transcription factor

whose activation might be influenced by cTPxI

It is worth noting the influence of the functional

state of mitochondria in the H2O2-induced expression

levels of various genes, at least in tsa1 mutant (Fig 4,

compare lanes 6 and 8), suggesting that

respiratory-compromised cells fail to activate some H2O2

respon-sive genes transcription at the same degree of

respirat-ory-competent ones The H2O2-induced expression

levels of GSH1, PRX1, CCP1, CTT1, TRR1 were

affected the most by the defective mitochondria, while

those of GPX2, TRX2, TRX3, SOD2 GRX5, POS5

were influenced at a lower level

The treatment with antimycin A alone, in all cases,

led to expression levels resembling those observed in

control cells (Fig 4) These results are in agreement

with genome-wide studies that did not find significant

differences in the expression of antioxidant genes in

cells with mitochondrial dysfunction [44,45]

Participation of transcription factors in the

antioxidant defense of cells with normal or

impaired mitochondrial function

In order to identify transcription factors involved in

the response of cells with dysfunctional mitochondria

to oxidative stress, we evaluated H2O2 sensitivity of deletion mutants for the regulators most frequently associated with oxidative stress response: Yap1, Skn7, Msn2 and Msn4

Single or double mutants for Yap1 and Skn7 were very sensitive to H2O2, although deletion of YAP1 gene appeared to be more deleterious than the SKN7 gene deletion (Fig 5) This high sensitivity was already expected given the diversity of antioxidant enzymes regulated by these factors [7] The association of H2O2 with antimycin A totally inhibited growth of these mutants In contrast, no further growth inhibition of yap1D and skn7D was achieved by the association of FCCP with H2O2, relative to H2O2 alone (Fig 5) No significant growth retardation of yap1D and skn7D rel-ative to wild-type counterparts was observed when these cells were treated with antimycin A alone or with FCCP alone Therefore, the phenotypes of yap1D and skn7D, were similar to those of tsa1D described in Fig 1, suggesting that Yap1, Skn7 and cTPxI act in the same pathway in the response of yeast with dys-functional mitochondria to oxidative stress Because cell growth was more affected by the deletion of YAP1 and SKN7 genes than by deletion of cTPxI (Fig 5),

we suggest that other enzymes whose genes are regula-ted by these factors could also be involved in the anti-oxidant defense of respiratory-incompetent cells

On the other hand, Msn2 and Msn4 appear to not play significant role in the response of cells to H2O2or

to either of the compounds that interfere with mitoch-ondrial function (antimycin A or FCCP), as no consid-erable growth alterations for their deletion mutants were detected (Fig 5) Since Ras-cAMP-PKA pathway inhibits Msn2⁄ 4 under catabolic repressing conditions [14], the sensitivity of msn2Dmsn4D was also evaluated

in the absence of glucose In this case, cells were grown

in raffinose medium Again, these mutants grew simi-larly to the wild-type cells (data not shown), dismissing the involvement of Msn2⁄ 4 in the response of respirat-ory incompetent cells to oxidative stress

yap1∆skn7 ∆

skn7∆

WT

yap1∆

msn2∆msn4 ∆

anti A

H2O2+ FCCP

Fig 5 Sensitivities of mutants lacking transcription factors to several stressful conditions Growth of the strains BY4741 (WT), and mutants indicated on YPD plates containing no chemicals as a control, 0.8 m M H2O2, 0.1 lgÆmL)1antimycin A (anti A), and 2.5 lgÆmL)1FCCP singly

or in association Proceedings were performed as described in Fig 1.

Yap1 cellular distribution

It is well known that Yap1 is accumulated in the

nuc-leus of cells exposed to oxidative stress and, as a

con-sequence, the expression of its target genes is activated

[11,46,47] We observed that mitochondrial dysfunction

negatively affects the H2O2-induced expression levels

of various Yap1-target genes in tsa1 mutant cells

(Fig 4, compare lanes 6 and 8), which could account

for the decreased capacity of yeast to cope oxidative

stress (Fig 1) To check this possibility, we examined

the distribution of Yap1 in the cells expressing

GFP-Yap1 fusion protein

No significant difference in the cellular GFP-Yap1

distribution was observed between the wild-type and

tsa1 mutant cells in all of the conditions tested

(Fig 6) In spite of the increased basal expression

lev-els of a variety of genes in tsa1 cells, we did not

observe GFP-Yap1 accumulation in the nucleus of

these cells, corroborating results previously obtained

[27] Therefore, GFP-Yap1 is located in nucleus and

cytoplasm of both wild type and tsa1D cells in control

conditions (Fig 6) Antimycin A treatment alone did

not lead to a nuclear accumulation of GFP-Yap1

(Fig 6), which is in agreement with the similar

expres-sion levels of genes from control and antimycin

A-trea-ted (Fig 4, compare lanes 1 with 3 and 5 with 7)

Antimycin A treatment did not alter the nuclear

Yap1 accumulation induced by H2O2, in neither the

wild-type nor in tsa1D cells (Fig 6) Hence, the

dimin-ished H2O2-induced expression levels of some Yap1-target genes observed in cells with impaired mitochondrial function can not be attributed to alter-ation in Yap1 cellular distribution Probably other fac-tors, such as ability of Yap1 to bind DNA [51], are also involved in the activation of genes involved in the response of yeast to oxidative stress These possibilities are further discussed below

Discussion

It was previously demonstrated that cTPxI is essential for the antioxidant defense of cells with mitochondrial dysfunction [35] Remarkably, we have shown here that cTPxI is very specific among several other antioxi-dants in the protection of cells with respiratory incom-petence against peroxides (Fig 1) The protective action of cTPxI was prominent in situations of elec-tron flow impediment This was demonstrated by the severe growth retardation (Fig 1) and by the large depletion of sulfydryl content (Fig 2) of tsa1 mutant treated with H2O2 in association with antimycin A, effects that were not observed when these cells were exposed to H2O2+ FCCP As it has long been shown, while antimycin A augments ROS generation by defective mitochondria [19–21], FCCP diminishes it [37–39] Therefore, it is possible that cTPxI could be specifically important when internal ROS production is elevated In agreement, it was demonstrated that a bacterial peroxiredoxin, alkyl hydroperoxide reductase

control

H2O2

Anti A

Anti A +H2O2

Fig 6 Cellular distribution of GFP-tagged

Yap1 Cells of the strains JD7–7C (WT) and

tsa1D carrying expression plasmids for the

GFP-YAP1 fusion gene were exposed to 1.2

m M H 2 O 2 and 0.1 lgÆmL)1antimycin A (anti

A), separately or in association, and confocal

laser scanning microscopy was carried out

as described under ‘Experimental

proce-dures’ The left panels show the fluorescent

images and the right panels show the

trans-mission images All of the data shown are

representative of at least three independent

experiments, all of which gave similar

results.

(AhpC), is the primary scavenger of endogenous H2O2

[48] Altered ATP levels do not appear to influence

res-piratory deficient yeast antioxidant defenses, since cells

treated with FCCP, which leads to a more pronounced

energy limitation due to the higher cytoplasmic ATP

hydrolysis rate [37] did not present alteration in H2O2

sensitivity, even for the tsa1 mutant (Fig 1)

The peroxidatic function of cTPxI probably overlaps

to some extent with other H2O2 detoxifying enzymes,

but the recent finding that this protein possesses

chap-erone activity under stressful conditions provides a

very tempting explanation for the distinctive role of

cTPxI in protecting cells with mitochondrial

dysfunc-tion against oxidative stress Indeed, our data showed

that cTPxI appears not only as low molecular weight,

but mostly as high molecular weight complexes in cells

exposed to H2O2 alone or in association with

antimy-cin A (Fig 3), suggesting that it may be acting as

per-oxidase and as chaperone under these conditions, as

this functional and structural correlation was already

well demonstrated [33,34] Its high molecular weight

form with chaperone activity could protect essential

proteins from denaturation or could mediate

activa-tion of downstream defense signaling cascades that

prevent H2O2-induced cell death Actually, it was

dem-onstrated that oxidant-mediated proper folding of

Yap1 is required for transcriptional activation and for

the nuclear accumulation of this regulator during

stress [49]

Another phenomenon that could be related to the

unique phenotype of tsa1 mutant cells is that most

thiol proteins are inactivated when oxidized to sulfinic

acids, because this oxidation state of cysteine residues

is not reducible by classical reducing agents such as

glutathione and thioredoxin In contrast, the sulfinic

acid form of cTPxI can be specifically reduced by

sul-firedoxin [36] In fact, srx1D presented similar

pheno-type of tsa1D cells (Fig 1) The fact that antimycin A,

but not diamide, increased the sensitivity of tsa1

mutant cells to peroxides (Fig 1) gave support to the

notion that higher oxidation states of cysteines might

be taking place in the cTPxI dependent response to

oxidative stress This is because diamide is an oxidant

that gives rise only to disulfides [40], whereas peroxides

generate disulfides as well as sulfenates (Cys-SOH),

sulfinates (Cys-SO2H) and sulfonates (Cys-SO3H) The

hypotheses raised here to explain the unique tsa1D

phenotype are not mutually exclusive Actually, sulfinic

acid formation in cTPxI by H2O2 was suggested as a

trigger event for the switch of this peroxiredoxin from

a peroxidase to a chaperone enzyme [33]

2-Cys Prxs have been implicated in the regulation of

stress-induced gene expression [27–32] A previous

work has shown that expression levels of GSH1, GLR1 and GPX2 were increased in a tsa1 mutant and this effect was dependent on Yap1, since transcriptional activation of these genes were not observed in tsa1D⁄ yap1D double mutants [27] Our data confirmed these results, but indicate that there may be more mecha-nisms involved in this TSA1-dependent regulation For some genes, the transcription increase seemed to be stress-independent, while for others, it was dependent

on H2O2 (Fig 4) It is most relevant that, in several cases, the H2O2induction was reduced by the presence

of antimycin A, suggesting that the functional state of the mitochondria is somehow sensed by regulatory mechanisms Perhaps the decreased levels of these enzymes, and other yet not detected, could be respon-sible for the reduced viability of tsa1D cells grown in the presence of H2O2 plus antimycin A (Fig 1) The existence of a connection between cTPxI and Yap1 is becoming evident, although its molecular basis

is not yet clear It was demonstrated that Yap1 is retained in the nucleus in the presence of H2O2 and thereby it interacts with the target genes [11,46,47] This retention is dependent of the oxidation of Yap1 cysteine residues by H2O2, that modifies its conforma-tion and hinders its interacconforma-tion with Crm1, which oth-erwise would export this factor to cytoplasm H2O2 oxidizes Yap1 in a process mediated by Gpx3⁄ Orp1, a glutathione peroxidase homologue with thioredoxin peroxidase activity [11], with a still unclear participa-tion of Ybp1 [50] Interestingly, it was demonstrated in cells with a truncated form of Ybp1, that cTPx1 can replace Gpx3⁄ Orp1 in the oxidation of Yap1 thus pro-moting its nuclear retention [30] Moreover, in Schizo-saccharomyces pombe, a 2-Cys Prx, but not a GPx, directly oxidizes Pap1 (a Yap1 homologue) provoking the nuclear retention of this transcription factor [51] Despite the observed alterations in the expression

of Yap1-target genes, the cellular localization of this transcription factor was not altered by either the TSA1 deletion or by the inhibition of mitochondrial function (Fig 6) Inoue et al [27] have also demon-strated that the expression of a reporter gene fused

to a Yap1-dependent promoter was significantly increased in the absence of cTPxI by a mechanism independent of Yap1 nuclear retention There is a precedent showing that Yap1 binding activity may be affected It was shown that the accessibility of Yap1

to the GSH1 promoter could be repressed by Cbf1, a DNA-binding protein that binds to elements in the vicinity of Yap1 binding site [52] Alternatively, the increased reducing power of tsa1 mutant, achieved by the expression elevation of GSH1, GSH2, GLR1 and TRR1 and detected by our analysis (Fig 2), could

contribute to the reduction of Yap1, hence

diminish-ing the oxidized Yap1 ‘lifetime’, thus avoiddiminish-ing its

accumulation in the nucleus In fact, Wiatrowski and

Carlson [53] did not observe Yap1 accumulation in

the nucleus of cells shifted from glucose to glycerol,

otherwise observed, in the presence of glutathione

externally added

Another striking point in the adaptation of cells

to H2O2 is the redirection of carbohydrate flux from

hexose phosphate pool (glycolysis) to the pentose

phosphate pathway to the regeneration of NADPH

[12] which, in turn, is responsible for the maintenance

of both thioredoxin and glutathione in their reduced

states As cells treated with antimycin A rely only on

glycolysis to produce ATP, the carbohydrate

meta-bolism redirection after H2O2 treatment would be

affected and the generation of NADPH would be

diminished The depletion of sulfhydryl groups

occurred in tsa1 cells treated with antimycin A plus

H2O2(Fig 2) could corroborate with this hypothesis

These multiple activities of cTPxI (peroxidase,

chap-erone and redox signaling) might be related to the

cen-tral roles of this protein in prevention of yeast against

genotoxic processes [54,55] Here, we have shown some

alterations that could impair the oxidative stress

response of yeast cells with mitochondrial dysfunction

and that cTPxI is specifically important in their

anti-oxidant defense Although the mitochondrial inhibition

procedures used here were extreme, these approaches

have been largely employed in bioenergetics studies and

have provided valuable information Moreover, the

nonphysiological doses of peroxides used here were due

to the high redundancy of the yeast antioxidant systems

Nevertheless, our results suggest that peroxiredoxins,

especially those with high similarity to the yeast cTPxI

could exert a decisive role in the establishment of

mitochondrial dysfunction-related diseases, although

further studies are necessary to ascertain this

relation-ship In support of this hypothesis, peroxiredoxins have

been implicated in the development of different kinds of

cancer [56–60] and neurodegenerative diseases [61,62]

Experimental procedures

Yeast strains and growth conditions

The following S cerevisiae strains were used in this study:

JD7–7C (MATa ura3–52 leu2 trpA K +) and tsa1D (MATa

ura3–52 leu2 trpA K + tsa1D::LEU2) were obtained from

Chae [63] (National Institute of Health, Bethseda, MD,

USA); BY4741 (MATa; his3D1; leu2D0; met15D0; ura3D0),

tsa1D (MATa; his3D1; leu2D0; met15D0; ura3D0; tsa1D::

Kan Mx4), prx1D (MATa; his3D1; leu2D0; met15D0;

ura3D0; prx1D::Kan Mx4) tsa2D (MATa; his3D1; leu2D0;

his3D1; leu2D0; met15D0; ura3D0; ahp1D::Kan Mx4), ccp1D (MATa; his3D1; leu2D0; met15D0; ura3D0; ccp1D::Kan Mx4), glr1D (MATa; his3D1; leu2D0; met15D0; ura3D0; glr1D::Kan Mx4), yap1D (MATa; his3D1; leu2D0; met15D0; ura3D0; yap1D::Kan Mx4), skn7D (MATa; his3D1; leu2D0; met15D0; ura3D0; skn7D::Kan Mx4) were obtained from

101 ura3–52), ctt1D (MATa trp-D1 his3-D200 lys2–801 leu2-D1 ade2–101 ctt1::URA3), cta1D (MATa his3-D200 lys2–

801 leu2-D1 ade2–101 ura3–52 cta1::TRP1) were obtained from Izawa [64] (Kyoto University, Japan), and W303–1a (MATa, ade2, can1, his3, leu2, trp1, ura3) and msn2Dmsn4D (MATa, ade2, can1, his3, leu2, trp1, ura3, msn2::HIS3, msn4::TRP1) were obtained from Boy-Marcotte [65] (Uni-versite Paris-Sud, France)

extract, 2% bacto-peptone, 2% glucose) For most analysis, cells were harvested by centrifugation at mid-log phase,

Determination of tolerance to different oxidants Spot test: cells were first grown in YPD media until a

these cell suspensions were realized and a 12 lL droplet of each was plated on YPD-agar medium containing 1.2 mm

associ-ation Plates were then incubated 2 days Only the three highest dilutions were represented in the figures

Determination of sulfhydryl groups PB-SH levels were measured according to the method of Sedlak and Lindsay [66], by subtracting the NP-SH content from the total sulfhydryl (T-SH) content Cells of the strains JD7–7C and tsa1D were grown on YPD and, after

cells from each culture were collected Protein extracts were obtained in 0.02 m EDTA pH 4.7 with glass beads addition followed by

centrifugation at 17 900 g for 15 min The T-SH

concentra-tions were determined by absorption levels at 412 nm after incubating 200 lL aliquots of protein extracts supernatants with 780 lL 0.2 m Tris pH 8.2 and 20 lL 5 mm DTNB for

30 min The NP-SH contents were determined in the super-natant, after proteins precipitation with 5% trichloroacetic acid (final concentration) by incubating 450 lL supernatant,

900 lL 0.4 m Tris pH 8.9 and 26 lL 5 mm DTNB for

5 min Absorption levels were measured at 412 nm

Determination of the switching of cTPxI

structures in vivo

Cells grown on YPD were treated during 40 min with

FCCP, separately or in association The corresponding

whole cell extracts, obtained as described by Ausubel et al

overnight running) and subjected to immunoblotting with

an anti-cTPxI antibody The nondenatured protein

molecu-lar weight marker kit was purchased from Sigma As

posit-ive control, recombinant cTPxI was also present in the gels

DNA manipulation

To generate the probes for northern blot analysis, the

DNA sequences of the selected antioxidant genes were PCR

Expression Clones, Research Genetics (Invitrogen,

Madi-son, WI, USA) The clones containing the expression

plas-mids corresponding to the ORFs of interest (YPL091W,

YJL101C, YML028W, YLR109W, YDR353W, YDR453C,

YDR513W, YNL241C, YCL035C, YFL039C, YHR008C,

YIL010W, YLR043C, YGR088W, YJR104C, YBL064C,

YDR256C, YDL066W, YPL188W, YOL049W, YAL005C,

YLL026W and YIR037W) were grown separately on YPD

medium, and DNA of each clone was extracted as

des-cribed by Ausubel et al [67] PCR was carried out using

the following primers: 5¢-GAATTCCAGCTGACCACC-3¢

PCR products were purified and the sequences were

con-firmed previously to the probes preparation

RNA isolation and analysis

Total yeast RNA was extracted by the method of hot acid

phenol method and northern blotting was performed as

pre-pared by random primed synthesis [67] Actin was used as

loading control and no significant difference was found

rel-ative to ribosomal RNA (not shown)

Localization of GFP-tagged Yap1

The expression plasmids for the green fluorescent protein

(GFP) fused to Yap1 were kindly provided by Kuge [68]

They were transferred to cells of the strains JD7–7C and

tsa1D Cells were grown on YPD to mid-log phase,

and 5 lL of each culture were spotted on to glass slides

Confocal laser scanning microscopy analysis was performed

using a Zeiss LSM510 Axiovert 200 m microscope (Carl

Zeiss MicroImaging, Inc., Thornwood, NY, USA) by

Acknowledgements

We thank Dr Shusuke Kuge for providing strains and plasmids We also thank Hugo Metz for technical assistance with the confocal laser scanning microscopy analysis and Lyndel Meinhardt for his comments on the manuscript Special thanks to Vasco dos Santos Dias (in memoriam) This work was supported by grants from the Brazilian Agencies FAPESP and CNPq

References

1 Hallywell B & Gutteridge JMC (1989) In Free Radicals

JMC, eds), 2nd edn Claredon Press, Oxford

2 Finkel T & Holbrook NJ (2000) Oxidants, oxidative stress and the biology of ageing Nature 408, 239–247

3 Jamieson DJ (1998) Oxidative stress responses of the yeast Saccharomyces cerevisiae Yeast 14, 1511–1527

4 Grant CM (2001) Role of the glutathione⁄ glutaredoxin and thioredoxin systems in yeast growth and response

to stress conditions Mol Microbiol 39, 533–541

5 Collinson EJ & Grant CM (2003) Role of yeast gluta-redoxins as glutathione S-transferases J Biol Chem 278, 22492–22497

6 Park SG, Cha MK, Jeong W & Kim IH (2000) Distinct physiological functions of thiol peroxidase isoenzymes

in Saccharomyces cerevisiae J Biol Chem 275, 5723– 5732

7 Lee J, Godon C, Lagniel G, Spector D, Garin J, Laba-rre J & Toledano MB (1999) Yap1 and Skn7 control two specialized oxidative stress regulons in yeast J Biol Chem 274, 16040–16046

8 Charizanis C, Juhnke H, Krems B & Entian K-D (1999) The mitochondrial cytochrome c peroxidase Ccp1 of Sacharomyces cerevisiae is involved in conveying an oxidative stress signal to the transcription factor Pos9 (Skn7) Mol Gen Genet 262, 437–447

9 Costa V & Moradas-Ferreira P (2001) Oxidative stress and signal transduction in Saccharomyces cerevisiae: insights into ageing, apoptosis and diseases Mol Aspects Med 22, 217–246

10 Moye-Rowley WS (2002) Transcription factors regula-ting the response to oxidative stress in yeast Antioxid Redox Signal 4, 123–140

11 Delaunay A, Pflieger D, Barrault M, Vinh J & Toledano

redox transducer in gene activation Cell 111, 471–481

12 Godon C, Lagniel G, Lee J, Buhler JM, Kieffer S, Per-rot M, Boucherie H, Toledano MB & Labarre J (1998)

Chem 273, 22480–22489

13 Hasan R, Leroy C, Isnard A-D, Labarre J, Boy-Mar-cotte E & Toledano MB (2002) The control of the yeast