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INTRODUCTION |
The incomplete reduction of molecular oxygen during respiration
and the lipid metabolism in peroxisomes leads to the formation of
reactive oxygen species
(ROS)1 (1). ROS are potent
oxidants and can damage all cellular constituents (2). They cause DNA
base modifications and strand breaks and are therefore mutagenic. They
can damage proteins and inactivate enzymes. Oxidation of membrane
lipids can initiate free radical chain reactions, which alter cellular
membranes and give rise to other very toxic reactive species such as
lipid hydroperoxides, alkoxyl and peroxyl radicals, lipid epoxides, and
aldehydes. To protect against the toxicity of ROS, aerobic organisms
use an array of defense mechanisms (1, 3, 4). Among these, cytosolic and mitochondrial superoxide dismutases eliminate the O
2
radical, cytosolic and peroxisomal catalases remove
H2O2, and glutathione peroxidases reduce both
H2O2 and alkyl hydroperoxides. The AhpC/TSA family, also referred to as peroxiredoxin, is a large family of newly
discovered peroxidases that are highly conserved from prokaryotes to
eukaryotes (5) and act to reduce hydroperoxides with electrons donated
by NADPH via thioredoxin or other thiol-containing intermediates (6-8). Antioxidant defense mechanisms are induced as part of global
cellular adaptive responses to oxidative stress (9-12). In
Saccharomyces cerevisiae, the cellular response to
hydroperoxides is associated with the induction of a very large
stimulon of at least 115 proteins (13), which involves, at least in
part, the transcriptional regulators Yap1 (14, 15) and Skn7 (16, 17). Strains inactivated for either one of these regulators are
hypersensitive to killing by H2O2 and by
several other oxidants (15, 16, 18). Yap1 controls the expression of
GSH1-encoded
-glutamylcysteine synthetase (19) and of
GLR1-encoded glutathione reductase (20) and co-operates with
Skn7 to activate the expression of TRX2-encoded thioredoxin
and TRR1-encoded thioredoxin reductase (17).
To gain insight into the molecular physiology of the yeast oxidative
stress response, we have attempted to identify new genes involved in
the cellular defense against alkyl hydroperoxides and possible targets
of Yap1 and Skn7. As an approach to this question, we have used a
genetic selection for identifying genes, which could rescue the alkyl
hydroperoxide hypersensivity of an skn7 null strain. We
report the identification of one of the genes isolated from this
selection, which we have named AHP1 (see below). Evidence is
provided which demonstrates that the AHP1 gene product is a
yeast antioxidant related to the AhpC/TSA peroxiredoxin family with
alkyl hydroperoxide defense properties.
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MATERIALS AND METHODS |
Yeast Strains and Growth Conditions
Studies were performed with the wild type strain YPH98 (21) as
indicated in the legends (MATa ura3-52
lys2-801amber
ade2-101ochre trp1-
1
leu2-
1) and its isogenic derivatives yMT1
(yap1::LEU2), yMT2
(skn7::TRP1), yMT3 (yap1::LEU2,
skn7::TRP1), yMT4 (gsh1::LEU2), yMT5 (ahp1::TRP1), yMT6
(tsa1::TRP1), and yMT7
(trr1::TRP1). The following strains were also used
as indicated in the legends and are derived from W303: wild type strain
EMY60 (MATa, ade3, ade2 trp1-1, leu2-3, 112, ura3-1, his3-11, can1-100), EMY61
(trx1::TRP1), EMY62
(trx2::LEU2), and EMY63
(trx1::TRP1, trx2::LEU2) and are a gifts
from Dr. E. G. Muller (University of Washington, Seattle, WA) (22,
23). The composition of synthetic complete medium (SC), rich broth
medium (YPD), glucose (SMG), and galactose selective media (SMGal) are
described elsewhere (24). Strains were transformed by electroporation
as described (25).
Gene Disruptions and Plasmid Constructions
Standard protocols and buffers were used (26). Gene disruptions
were performed by the one-step gene disruption technique (25). The
skn7::TRP1 construct used to create yMT2 was a
gift from Dr. H. Bussey (McGill University, Montreal, Canada) (27). The
yap1::LEU2 construct used to create yMT1 was
prepared by removing the YAP1 coding sequence from the
BamHI site (+186) to the KpnI site (+1650)
relative to the ATG and replacing it with the LEU2 gene. The
ahp1::TRP1 deletion construct used to generate
yMT5 was prepared by replacing the full AHP1 open reading
frame (ORF) with the TRP1 gene. Basically, oligonucleotide
primers were used to PCR-amplify 5' (
680 to +5) and 3' (+532 to
+1530) regions of the AHP1 gene relative to the ATG. These
PCR-amplified fragments were then subcloned upstream and downstream of
the TRP1 gene. The deletion construct used to create yMT4
(gsh1::LEU2) was prepared by removing a 700-base
pair DNA fragment from the HindIII site at
180 base pairs
relative to the ATG up to the BamHI site at +511 and
replacing it with the LEU2 gene. yMT6 and yMT7 were prepared by a one-step amplification protocol that replaced the entire TSA1 or TRR1 ORF with the TRP1 gene
(28). The cDNA library plasmid pY1 contained the entire
AHP1 ORF from
119 relative to the translation start to
+614, 82 base pairs downstream of the stop codon. To generate
pAHP1, the AHP1 gene was isolated by PCR
amplification of total yeast DNA with oligonucleotides that primed from
984 to +614, and then cloned into the polylinker region of plasmid pRS426 between the BamHI and NotI sites.
Selection of Yeast cDNAs
A S. cerevisiae cDNA library under control of the
repressible GAL1 promoter in the centromeric vector pRS316
(29) was transformed into strain yMT2
(skn7::TRP1). Transformed cells were plated onto SMG lacking uracil and containing 1 M sorbitol.
URA3 transformants were pooled from each plate separately,
diluted, and then plated onto SMGal lacking uracil and containing
t-BOOH (0.5 mM). Plates were incubated at 30 °C for
5-12 days. Plasmids recovered from viable colonies were analyzed by
restriction digest and sequencing. Sequencing was performed using an
automated Perkin-Elmer DNA sequencer according to the recommendations
of the manufacturer.
Sensitivity Assays
Patch Assays--
Aliquots (10 µl) containing approximately
103 or the indicated cell number of an overnight culture
were spotted on YPD plates containing oxidants at the indicated
concentration. Plates were monitored after 3-6 days of incubation at
30 °C.
Challenge Assay--
Cells from an exponential growth culture
were washed, resuspended in phosphate-buffered saline at a density of
OD600 of 0.1, and not exposed or exposed to peroxides at
different concentrations for 1 h. Cells were then diluted and
plated for colony survival.
Northern Blot Analysis
Yeast cells from overnight culture were diluted to an
OD600 of 0.01 in SC medium and incubated with shaking at
30 °C until they reached an OD of 0.3. The cells were then aliquoted
and incubated in the absence or in the presence of
H2O2 (0.2 mM) or t-BOOH (0.2 mM) for 20 min. Total RNA was prepared by the hot phenol
method (30). For each condition tested, 20-µg RNA samples were loaded per lane on an agarose gel containing formaldehyde, separated by
electrophoresis, transferred to a Bio-Rad nylon membrane, and hybridized with an an AHP1 32P-labeled DNA
specific probe produced by random priming (Boehringer Mannheim).
Hybridization of each blot with a small nuclear RNA U3
(SNR17A)-specific 32P-labeled DNA probe served as a RNA
loading control. Prehybridization, hybridization, and washes were
carried out as described elsewhere (26). Hybridized membrane were
exposed for autoradiography.
Two-dimensional Gel Electrophoresis and Protein Spot
Identification
Two-dimensional gel electrophoresis and protein spot
identification were performed as described elsewhere (13). Alkylation of protein free sulfhydryls before the second dimension was achieved as
follows. The first dimensional gel rod was first reduced by a 10-min
incubation in buffer A (30% glycerol, 6 M urea, 2.5% SDS,
0.001% bromphenol blue, 0.2 M Bis-Tris, 0.1 M
HCl, and 67 mM dithiothreitol (DTT)). Alkylation was then
performed by incubating the gel rod in buffer in which DTT was omitted,
and iodoacetamide (222 mM) was added instead.
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RESULTS |
Selection of Genes Increasing the Tolerance to t-BOOH of a skn7
Null Strain--
A S. cerevisiae strain carrying a
skn7 null mutation fails to grow on medium containing 0.5 mM of t-BOOH (a synthetic alkyl hydroperoxide), whereas the
isogenic wild type strain will tolerate up to 1.5 mM of
this oxidant. We have searched for genes whose overexpression would
rescue the t-BOOH hypersensitivity of the skn7 null strain
using a galactose-inducible yeast cDNA library (29). This library
was used to bypass the possible Skn7 requirement for the expression of
candidate genes. We expected to identify genes controlled by Skn7
and/or encoding alkyl hydroperoxide antioxidant defense activities.
Strain yMT2 (skn7::TRP1) was transformed with the
GAL1-controlled cDNA library. Forty thousand
transformants were obtained and incubated on plates containing 0.5 mM t-BOOH. Viable colonies were recovered from these plates
after 7 days and the corresponding library plasmids isolated. Five
plasmids, able to increase the t-BOOH tolerance of the yMT2 strain upon retransformation, were shown to carry inserts of different sizes, but
containing the same ORF. One of them, pY1, was further analyzed. pY1
was able to increase the tolerance to t-BOOH not only of yMT2 but also
of yMT1 (a yap1 null strain) (Fig.
1). Therefore, the gene carried on the
pY1 plasmid has antioxidant properties. This gene was called
AHP1 for alkyl hydroperoxide reductase (see below) and
further analyzed. The AHP1 gene is encoded at the YLR109W locus. Interestingly, its gene product has been recently identified as
a 19-kDa protein by comparative two-dimensional gel electrophoresis in
the search of yeast proteins induced by H2O2
(13).

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Fig. 1.
The AHP1 gene partially
rescues the t-BOOH-hypersensitive phenotype of skn7
and yap1 null strains. skn7 and
yap1 null strains not carrying ( skn7,
yap1) or carrying the pY1 plasmid
( skn7pGAL1AHP1,
yap1pGAL1AHP1), which expresses the
AHP1 gene under control of the GAL1 promoter and
isogenic wild type strain, were compared by the patch assay for their
ability to grow on SDGal solid medium containing t-BOOH at the
indicated concentration. For each strain, 10 µl of an overnight
culture (OD600 2-3) diluted to 20, 5, or 1 × 103 cells (from left to right) were
spotted on tester plates.
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Similarity of Ahp1p with Peroxisomal-like Proteins and with the
AhpC/TSA Protein Family--
Ahp1p is a protein predicted to be 176 amino acids long with a molecular mass of 19,108 Da (Fig.
2). This predicted mass is in agreement
with a molecular weight of 19,000 extrapolated from its mobility in
denaturing polyacrylamide gels (13) (see also Fig. 6). Ahp1p contains 3 cysteine residues at positions 31 (Cys-31), 62 (Cys-62), and 120 (Cys-120). Ahp1p carries the C-terminal sequence AHL similar to the
putative S(A)K(H/R)L(I) peroxisomal-like sorting signal sequence (31).
A sequence comparison with the GenBank data base revealed that Ahp1p is
highly similar throughout its entire sequence to five peroxisomal-like
fungal proteins, and to three other prokaryotic proteins. Ahp1p has
also a weak, yet significant similarity to the peroxiredoxin AhpC/TSA
family.

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Fig. 2.
Nucleotide sequence of the AHP1
gene and the predicted amino acid sequence of the Ahp1
protein. The nucleotide sequence of the AHP1 gene and
its flanking regions is shown in the 5' to 3' direction. The predicted
amino acid sequence of Ahp1p is indicated below the relevant
nucleotides. The Yap1 response element (TTAGTAA) and the three putative
stress response elements (CCCCT) are indicated in boldface
and underlined. The two TATA boxes are also indicated. The
three cysteines and the AHL peroxisomal-like sorting signal of Ahp1p
are indicated in boldface.
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The sequences of the proteins that are highly similar to Ahp1p are
compared in Fig. 3. Of the three Ahp1p
cysteines, Cys-62 is the only one conserved among these related
proteins and occupies a region with a high density of amino acid
identities. Cys-31 is conserved only in Lipomyces
kononenkoae peroxisomal-like protein and in Aspergillus
fumigatus Aspf3 protein. C. bondinii PMP20 A and
B proteins (32) and the other related fungal protein sequences all
contain a tripeptide peroxisomal-like sorting signal at their extreme C
terminus. Inspection of these related sequences also shows that the
Haemophilus influenzae Y572 protein has an additional C-terminal domain with a very strong similarity to the redox-active center of glutaredoxins (Fig. 3). C. bondinii PMP20 A and B
are two isoforms of a protein that was identified as the most abundant peroxisomal proteins of methanol-exposed cells but was not assigned any
specific function (32, 33). None of the other proteins shown in Fig. 3
have an identified function. Nevertheless, the presence of a
glutaredoxin-like domain in Y572 suggests that this protein might be
involved in redox reactions.

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Fig. 3.
Sequence similarity between Ahp1p, a family
of peroxisomal membrane proteins, and the peroxiredoxin AhpC/TSA
family. Amino acid sequence alignment of Ahp1p with its fungi and
prokaryotic structural homologues. The alignment was performed using
the BLAST 2.0 at the National Center for Biotechnology Information
(47). Identical amino acids are boxed. Abbreviations and
percent of overall identity and similarity (in parentheses): L.k.
PLP, putative 17.3-kDa L. kononenkoae peroxisomal-like
protein (40%, 57%) (48); PMP20 A, C. bondinii
peroxisomal membrane protein A (39%, 56%); A.f.
Aspf3, A. fumigatus 18.5-kDa peroxisomal-like protein
(36%, 53%) (49); S.p. PMP20, Schizosaccharomyces
pombe PMP20 homologue (29%, 50%) (EMBL accession no. SPAJ2536);
Y572, hypothetical H. influenzae 26.7-kDa protein
(34%, 55%)(Swiss-Prot accession P44758); R.e. rpoN2,
Rhizobium etli hypothetical protein (29%, 50%) (EMBL
accession no. RET5696); S.sp. MP, Synechocystis
sp. hypothetical membrane protein (27%, 46%) (DDBJ accession no.
D90909).
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The overall similarity between Ahp1p and the AhpC/TSA family is
confined in a very short sequence surrounding Ahp1p Cys-62 and the
highly conserved N-terminal catalytic cysteine of the AhpC/TSA homology
(5, 7). This similarity between Ahp1p and the AhpC/TSA family is shared
among all the Ahp1p homologues shown in Fig. 3. The functional
importance of the N-terminal cysteine in the peroxidase function of
Tsa1p (7, 34) strongly suggests that Ahp1p and its homologues may have
an equivalent function. We therefore analyzed in detail the in
vivo antioxidant properties of Ahp1p.
Ahp1p Is an Antioxidant Specific for Organic Peroxides--
A null
mutation of the AHP1 gene that removes all its coding
sequence was created. The viability of the resulting ahp1
null strain demonstrates that this gene is not essential. The
ahp1 null strain does not have any particular growth
phenotype on glucose, galactose, acetate, pyruvate, or oleate media or
under anaerobic conditions. The effect of the ahp1 null
mutation was then tested on the stress tolerance to t-BOOH by a patch
assay. Compared with its isogenic wild type control, the
ahp1 null strain is hypersensitive to t-BOOH (Fig.
4A). Conversely, high
expression of AHP1 from a multicopy plasmid dramatically
increases the resistance of the wild type strain toward t-BOOH (Fig.
4C). Note that the difference in t-BOOH sensitivity of wild
type cells in panels A and C is related to the different strain backgrounds used in these experiments (see figure legend and "Experimental Procedures"). The role of AHP1 in the stress tolerance to H2O2
was then examined. In sharp contrast to its role in t-BOOH tolerance,
the ahp1 null mutation barely affects the cell tolerance to
H2O2 (Fig. 4B) and high expression of AHP1 does not improve the wild type strain tolerance to
H2O2 (Fig. 4C). Similar results were
obtained by a challenge assay (data not shown). These results suggested
that AHP1 is involved in the cellular defense against t-BOOH
but not against H2O2. This prompted us to
compare the antioxidant properties of Ahp1p with those of the
structurally related Tsa1p/peroxiredoxin. An in vitro measurement of Tsa1p catalytic activity had shown that this enzyme is
more efficient in reducing H2O2 than t-BOOH
(7). Significantly, the tsa1 null mutant is hypersensitive
to killing by H2O2 and has a wild type
resistance to t-BOOH (Fig. 4, A and B).

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Fig. 4.
Antioxidant and metal protective properties
of Ahp1p. A and B, ahp1 null
( ahp1), tsa1 null ( tsa1), and
isogenic wild type strains (WT) in the YPH98 background were
compared for their ability to grow on YPD not containing any added
chemical or containing either t-BOOH (A) or
H2O2 (B) at the indicated
concentrations. C, a wild type strain (EMY60) not carrying
( ) or carrying (+) an episomal plasmid expressing
AHP1 under control of its own promoter (pAHP1)
were tested for resistance to t-BOOH or to H2O2
at the indicated concentrations. D, an ahp1 null
( ahp1) and isogenic wild type strain (WT)
(YPH98) not carrying or carrying an episomal plasmid expressing
AHP1 under control of its own promoter (pAHP1)
were compared for their ability to grow on YPD containing diamide (1.4 mM), menadione (0.1 mM), or cadmium (0.1 mM). For each strain, 10 µl of an overnight culture
(OD600 2-3) diluted to 20, 5, or 1 × 103
cells (from left to right) were spotted on tester
plates. Growth was monitored after 3 days for all plates except for
cadmium plates and after 6 days for cadmium plates.
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The antioxidant properties of AHP1 were also tested toward
several other oxidants and a metal (Fig. 4D). The
ahp1 null mutation results in a slight decrease in the
tolerance to the thioloxidant diamide and to cadmium and suprisingly to
an increase in the resistance to the superoxide generating redox
cycling drug menadione. However, high copy expression of
AHP1 has no effect on the wild type strain tolerance to any
of these compounds.
The Antioxidant Function of Ahp1p Is Independent of GSH and
Dependent upon the Thioredoxin System--
Tsa1p1 reduces peroxides
with electrons donated by thioredoxin, thioredoxin reductase, and NADPH
(7, 34), whereas AhpC reduces organic peroxides with electrons donated
by the FAD-bound NADPH-dependent AhpF reductase (6). We
thus sought to determine the potential requirement of an electron donor
system for the Ahp1p antioxidant function. The effect of overexpressing
AHP1 on t-BOOH resistance was tested in strains deleted of
thioredoxin 1 (TRX1), thioredoxin 2 (TRX2), or
both TRX1 and TRX2 (Fig.
5A). The trx1 null
strain has a normal tolerance toward t-BOOH, whereas the
trx2 null mutant is slightly more sensitive than the wild type strain to this oxidant (15) (compare Fig. 5A with Fig. 4C). However, the trx1trx2 double null strain is
hypersensitive to t-BOOH. In the trx1 null strain,
overexpression of AHP1 results in a significant increase in
the resistance to t-BOOH, similar to that seen in the wild type strain
(compare Fig. 5A and 4C). In the trx2
null strain, the effect of overexpressing AHP1 can be seen
only at the lower t-BOOH concentrations. In the trx1trx2 null strain, no effect can be seen upon overexpression of
AHP1. A thioredoxin reductase (TRR1) deletion
mutant also displayed a t-BOOH-hypersensitive phenotype that was not
improved by overexpression of AHP1 (data not shown).

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Fig. 5.
The antioxidant function of Ahp1p is
independent of GSH but dependent upon the thioredoxin system.
Patch assays monitoring the sensitivity of genetically modified strains
to peroxides. A, isogenic strains, derived from EMY60, with
null deletions of TRX1 ( trx1), TRX2
( trx2), or both TRX1 and TRX2
( trx1 trx2), not carrying ( ) or carrying
(+) an episomal plasmid expressing AHP1 from its
own promoter (pAHP1) were tested on YPD solid media
containing t-BOOH at the indicated concentrations. B, a wild
type (WT) in the YPH98 background and isogenic strain with a
GSH1 null deletion ( gsh1) not carrying or
carrying an episomal plasmid expressing AHP1 from its own
promoter (pAHP1) were tested on YPD medium containing t-BOOH
at the indicated concentrations.
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The effect of overexpressing AHP1 on t-BOOH resistance was
also tested in strains deleted for
-glutamylcysteine synthase (GSH1), the rate-limiting enzyme in glutathione (GSH)
biosynthesis (Fig. 5B). The gsh1 null strain
appears slightly more resistant to t-BOOH than the wild type strain,
suggesting that high levels of GSH are not required for the resistance
to this oxidant (the gsh1 null strain is auxotroph for GSH
and can only grow in the presence of at least minimal amounts of
exogenous GSH supplement such as those present in YPD-rich medium; Ref.
35). In addition, the gsh1 null mutation does not affect the
gain in t-BOOH resistance resulting from AHP1
overexpression. Taken together, these data show that thioredoxins but
not GSH are important in the protection against t-BOOH and provide
indirect evidence for the requirement of the thioredoxin system, but
not of GSH, in the antioxidant protective function of Ahp1p.
Ahp1p Can Form a Disulfide-linked Homodimer in Vitro--
Ahp1p
was identified on two-dimensional gel electrophoresis of total yeast
proteins as a H2O2-inducible spot migrating at a pI of 4.5 and a molecular weight of 19,000. Interestingly, an unidentified H2O2-induced spot with a pI
identical to that of Ahp1p and a molecular weight of 40,000 was
observed (Fig. 6). We suspected that this
unknown spot could represent a dimeric form or other covalent
modifications of Ahp1p. This spot was indeed identified as Ahp1p by
peptide mapping using matrix-assisted laser desorption ionization-time
of flight/mass spectrometry (36). We next asked whether the 40-kDa spot
could represent a disulfide-linked dimer of Ahp1p. Cellular extracts
subjected to two-dimensional gel electrophoresis are initially reduced
in a buffer containing 160 mM
-mercaptoethanol. However,
some substrates could be potentially reoxidized during the second
dimension, when all of the
-mercaptoethanol has been lost during
electrophoresis. To test this hypothesis, the first dimension
polyacrylamide rod was incubated in a buffer containing DTT to reduce
any potentially reoxidized cysteine residues, and then treated in a
buffer containing the cysteine alkylating agent iodoacetamide, which
irreversibly blocks free sulfhydryls. As shown in Fig. 6, this
procedure led to the complete disappearance of the 40-kDa spot. These
data strongly suggest that the 40-kDa Ahp1p spot represents a
disulfide-linked Ahp1p homodimer.

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Fig. 6.
Ahp1p can form a disulfide-linked homodimer
in vitro. Autoradiogram of two-dimensional gel
electrophoresis performed with total cellular extracts from
[5S]methionine-labeled exponentially growing wild type
YPH98 cells. The 19-kDa Ahp1p protein and the 40-kDa Ahp1p dimer
identified by mass spectrometry are indicated by an arrow.
A, extracts prepared from untreated cells. B,
extracts prepared from cells exposed to H2O2
(0.4 mM) for 15 min. C, alkylated extracts
prepared from untreated cells. In this case, the two-dimensional gel
electrophoresis procedure was modified by the following change; after
the first-dimensional electrophoresis, the resulting polyacrylamide rod
was incubated in a buffer containing DTT and then treated with
iodoacetamide before subjecting it to the second electrophoretic
dimension.
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AHP1 Is Regulated by Yap1 and Skn7--
Ahp1p is an abundant
protein under non induced conditions, and its synthesis rate is
increased by about 3-fold upon exposure to H2O2
(13). We therefore analyzed whether the peroxide stimulation of Ahp1p
was dependent upon Yap1 and Skn7. Northern blots were performed using
an AHP1 specific probe and total RNA from exponentially growing wild type, yap1, and skn7 null cells that
had not been treated or were treated with 0.2 mM amounts of
either t-BOOH or H2O2. In wild type cells,
AHP1 mRNA levels were significantly increased upon
H2O2 (Fig.
7A) or t-BOOH (Fig.
7B) exposure, confirming the results of the two-dimensional
gel analysis. In contrast, in yap1 or in skn7
null strains, both uninduced and t-BOOH-induced AHP1
mRNA levels were significantly decreased, although they could still
be moderately induced by peroxide. However, this residual induction was
not seen in the yap1skn7 double null strain, although some
uninduced levels persisted (Fig. 7C). Consistent with a
transcriptional control by Yap1, the 5' AHP1 flank contains
the sequence TTAGTAA at position (
484) from the start codon, which
perfectly matches a Yap1 response element (Fig. 1) (37).

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Fig. 7.
AHP1 is an oxidative stress-inducible gene
regulated by Yap1 and Skn7. Northern blot analysis of
AHP1 transcription. A, total RNA was isolated
from exponentially growing (OD600 0.3) wild type cells
(WT), and isogenic strains with null deletion of
YAP1 ( yap1), SKN7
( skn7), which had not been treated or were treated for 20 min with H2O2 (0.2 mM) as
indicated. The resulting RNA were subjected to Northern blot which was
probed for AHP1 and U3 as a loading control, as
described under "Experimental Procedures." B, same as in
A, but cells were induced by t-BOOH (0.2 mM)
instead of H2O2. C,total RNA was
isolated from wild type (lane 1 to 3) and from an isogenic strain with
a null deletion of Yap1 and Skn7
( yap1 skn7). Cells were not treated
(lanes 1 and 4) or were treated for 20 min by H2O2 (0.2 mM)
(lanes 2 and 5) or by t-BOOH (0.2 mM) (lanes 3 and 6).
Densitometric quantification of the autoradiogram normalized to the
U3 loading control; relative values are given for each lane:
1 (2.83), 2 (8.16), 3 (6.37),
4 (0), 5 (1.68), and 6 (1.77).
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DISCUSSION |
Oxidative damage to lipids can lead to a variety of alkyl
hydroperoxides, which are particularly detrimental due to their ability
to initiate and propagate free radical chain reactions. Enzymes
involved in the breakdown of alkyl hydroperoxides are thus important
for protection against oxidative stress. We report here the
characterization of AHP1, which encodes a yeast antioxidant important for the protection against alkyl hydroperoxides, and related
to the AhpC/TSA peroxiredoxin family.
Gene deletion and overexpression analyses have demonstrated that
AHP1 exerts strong antioxidant protective functions.
Furthermore, the observation that AHP1 mRNA and protein
levels are induced by peroxides in a Yap1- and
Skn7-dependent manner also supports the notion that Ahp1p
is involved in protecting yeast cells from oxidative stress. Therefore,
AHP1 can be added to the growing list of yeast antioxidant
genes of the Yap1 and Skn7 regulons, which includes
TRX2-encoded thioredoxin (15), GSH1-encoded
-glutamylcysteine synthetase (19), GLR1-encoded
glutathione reductase (20), and TRR1-encoded thioredoxin
reductase (17).
Sequence analysis relates this antioxidant to the AhpC/TSA protein
family. Although mostly confined to a small region surrounding Cys62 of
Ahp1p and the highly conserved N-terminal cysteine of the AhpC/TSA
proteins family (5), this homology is functionally important because
the N-terminal cysteine of yeast Tsa1p is the catalytic center of its
peroxidase function (7, 34). The proposed catalytic mechanism for Tsa1p
involves substrate peroxide reduction by Cys N terminus, which upon
oxidation reacts with the Cys C terminus of another subunit to form an
intermolecular disulfide. This disulfide is subsequently reduced with
electrons donated by thioredoxin, thioredoxin reductase, and NADPH (7, 34). It was observed that Ahp1p forms a disulfide-linked homodimer upon
oxidation. Furthermore, genetic data suggested that the antioxidant protective function of AHP1 is independent of GSH but
dependent upon both thioredoxin and thioredoxin reductase. Therefore,
the structural similarity between Ahp1p and Tsa1p may be extended to a
common mode of action, which suggests that a peroxidase function underlies the antioxidant properties of Ahp1p. Of the three Ahp1p cysteines, Cys-62 would be most likely to be involved in direct substrate peroxide reduction. However, it is not clear whether another
cysteine would play a role in the putative Ahp1p catalytic function,
nor which cysteine(s) is (are) involved in homodimer disulfide bond
formation. Site-directed mutagenesis of individual cysteines of Ahp1p
will answer these questions.
The antioxidant protective function of AHP1 appears specific
for organic peroxides. The slight increase in
H2O2 sensitivity, and perhaps the altered
diamide tolerance phenotype seen in the ahp1 null mutant,
could be related to the lipid hydroperoxides which might be generated
upon exposure to these oxidants. Although not an oxidant, cadmium may
also lead to the production of ROS through metal-catalyzed electron
transfer (38, 39), and its toxicity might therefore be exacerbated by
the lack of AHP1. It is unlikely that cadmium is a direct
substrate for Ahp1p. The menadione hyperresistance phenotype of the
ahp1 null strain is not understood but could be related to a
deregulated expression of superoxide defense genes. Interestingly, an
in vivo comparison of the substrate specificities of Ahp1p
and Tsa1p showed that, whereas Ahp1p activity is specific for organic
peroxides, Tsa1p activity is specific for H2O2.
This observed in vivo substrate specificity of Tsa1p is
fully consistent with in vitro measurements of its catalytic
activity toward t-BOOH and H2O2 (7). Therefore, with respect to its substrate specificity, Ahp1p differs from Tsa1p and
appears related to the Escherichia coli and Salmonella typhimurium alkyl hydroperoxide reductase AhpC, which is
specifically involved in the breakdown of lipid and other alkyl
hydroperoxides, but not of H2O2 (6).
Eukaryotic cells have several enzymes involved in the breakdown of
lipid hydroperoxides. The structurally related selenoproteins glutathione peroxidase (40) and phospholipid hydroperoxide glutathione peroxidase (41, 42) can reduce both alkyl hydroperoxides and H2O2. However, glutathione peroxidase is more
active toward H2O2, whereas phospholipid
hydroperoxide glutathione peroxidase is more specific for lipid
hydroperoxides (41, 43). Furthermore, only phospholipid hydroperoxide
glutathione peroxidase can catalyze the reduction of lipid
hydroperoxide derivatives of intact phospholipids. Glutathione
S-transferase isozymes are also able to reduce lipid hydroperoxides with GSH (44, 45). Another pathway for the reduction of
lipid hydroperoxides is constituted by NADPH-dependent thioredoxin reductase (46). However, an eukaryotic orthologue of
prokaryotic AhpC has not been described. We suggest, based on substrate
specificity and structural homology, that Ahp1p is a peroxidase and the
yeast orthologue of prokaryotic AhpC.
Ahp1p is highly similar to C. bondinii PMP20 A and B and to
several other fungi and prokaryotic proteins (see Fig. 1B).
C. bondinii PMP20 A and B are two isoforms of a protein
identified as the most abundant peroxisomal protein of methanol-exposed
cells and suspected to play a role in methanol metabolism (32, 33). However, these two proteins and their fungi and prokaryotic homologues (see Fig. 3A), which share with Ahp1p the same similarity to
the AhpC/TSA family, may have also a peroxidase function. Consistent with this idea is the presence of a C-terminal glutaredoxin domain in
the H. influenzae Y572, which may serve as an electron donor for the catalytic cysteine of this putative peroxidase. Based on
immunostaining and cellular subfractionation experiments, PMP20 is a
peroxisomal protein tightly associated with membranes (33). Consistent
with this localization, PMP20 A and B contain a C-terminal peroxisomal
sorting signal sequence (32). A similar peroxisomal sorting signal is
present in Ahp1p and in the other PMP20-related fungi proteins,
suggesting that they may all be similarly localized in peroxisomes.
However, the abundance of Ahp1p observed on two-dimensional gels, which
only analyze soluble proteins, and its presence in high quantity in the
supernatant of a stationary cell culture (data not shown), may indicate
that this polypeptide is also present in the cytosol and is shed out of
the cell in the medium. A detailed analysis of Ahp1p subcellular
location will provide an answer to this question.
In summary, AHP1 encodes an AhpC/TSA-related antioxidant
with apparent substrate specificity for alkyl hydroperoxides. These data suggest that this antioxidant is a yeast orthologue of prokaryotic AhpC and justifies its name of Ahp1p for yeast alkyl
hydroperoxide reductase. AHP1 is
coordinately controlled with TRX2 and TRR1 by
Yap1 and Skn7 and requires the presence of these two enzymes to exert
in vivo its antioxidant protective function. In
vitro measurements of the antioxidant activity of wild type and
Ahp1p cysteine substitution mutants will provide a detailed analysis of
its peroxidase function and will confirm the role of the thioredoxin system in supporting the antioxidant activity of Ahp1p. Further studies
aimed at analyzing the redox characteristics of Ahp1p and understanding
the molecular basis of its substrate specificity will be of interest.
In that respect, the propensity of Ahp1p to oxidize to a
disulfide-linked homodimer during electrophoresis is quite striking and
suggests a high reactivity to oxidation in vitro and
probably also in vivo of one or more of its cysteines.