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Molecular and Cellular Biology, April 1999, p. 2650-2656, Vol. 19, No. 4
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Differential Protein S-Thiolation of
Glyceraldehyde-3-Phosphate Dehydrogenase Isoenzymes Influences
Sensitivity to Oxidative Stress
Chris M.
Grant,1,*
Kathryn A.
Quinn,2 and
Ian W.
Dawes1
Cooperative Research Center for Food Industry
Innovation, School of Biochemistry & Molecular
Genetics,1 and Center for Thrombosis
and Vascular Research, School of Pathology,2
University of New South Wales, Sydney, Australia
Received 25 September 1998/Returned for modification 11 November
1998/Accepted 21 December 1998
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ABSTRACT |
The irreversible oxidation of cysteine residues can be prevented by
protein S-thiolation, in which protein -SH groups form mixed disulfides
with low-molecular-weight thiols such as glutathione. We report here
the identification of glyceraldehyde-3-phosphate dehydrogenase as the major target of protein S-thiolation
following treatment with hydrogen peroxide in the yeast
Saccharomyces cerevisiae. Our studies reveal that this
process is tightly regulated, since, surprisingly,
despite a high degree of sequence homology (98% similarity and
96% identity), the Tdh3 but not the Tdh2 isoenzyme was
S-thiolated. The glyceraldehyde-3-phosphate dehydrogenase enzyme
activity of both the Tdh2 and Tdh3 isoenzymes was decreased following
exposure to H2O2, but only Tdh3
activity was restored within a 2-h recovery period. This indicates
that the inhibition of the S-thiolated Tdh3 polypeptide was
readily reversible. Moreover, mutants lacking TDH3 were
sensitive to a challenge with a lethal dose of
H2O2, indicating that the S-thiolated Tdh3
polypeptide is required for survival during conditions of
oxidative stress. In contrast, a requirement for the nonthiolated Tdh2
polypeptide was found during exposure to continuous low levels of
oxidants, conditions where the Tdh3 polypeptide would be S-thiolated
and hence inactivated. We propose a model in which both enzymes are required during conditions of oxidative stress but play complementary roles depending on their ability to undergo S-thiolation.
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INTRODUCTION |
Sulfhydryl groups (-SH) play a
remarkably broad range of roles in the cell, since the redox status of
cysteine residues is involved in both the structure and function of
numerous enzymes, receptors, and transcription factors. However,
cysteine residues are among the most easily oxidized residues in
proteins, and the oxidation of -SH groups is one of the earliest
observable events during reactive oxygen species (ROS)-mediated damage
(6, 12). The oxidation of -SH groups can result in the
formation of protein disulfides (S-S) through two protein cysteines or
mixed disulfides between protein-SH groups and a number of
low-molecular-weight thiols (protein S-thiolation). While the role of
disulfide bond formation in protein folding has been well characterized
(see, for example, reference 23), its role during
conditions of oxidative stress is unclear. In contrast, protein
S-thiolation has been proposed to serve an antioxidant function by
preventing the irreversible oxidation of cysteine residues to higher
oxidation states (e.g., sulfenic acid) following exposure to ROS
(10, 47).
Modification of proteins by S-thiolation does not require an enzymatic
activity and has been proposed to occur either by the reaction of
partially oxidized protein sulfhydryls (thiyl radical or sulfenic acid
intermediates) with thiols such as cysteine or glutathione (GSH)
or by thiol-disulfide exchange reactions with the oxidized disulfide
form of GSH (GSSG) (47). To provide a defense against
conditions of oxidative stress, this modification must be
reversible. Dethiolation can occur via direct reduction by GSH. In
addition, dethiolation can be catalyzed by glutaredoxin, thioredoxin,
and protein disulfide isomerase, but glutaredoxin appears to be the
most efficient dethiolase based on in vitro experiments
(29). The range of proteins that can be modified by
S-thiolation has been determined in studies with human endothelial cells and murine macrophages (40, 41). A few proteins that are S-thiolated in response to oxidants, including carbonic
anhydrase III (4), actin (3), creatine kinase
(8), glycogen phosphorylase b (37) and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (39,
42), have been positively identified in mammalian cells. The physiological relevance of the modification of these proteins is
unclear. There do not appear to be any unifying structural or
functional features apart from the relative abundance of these proteins
in mammalian cells.
The intracellular milieu is usually a reducing environment due to a
high concentrations (millimolar) of the low-molecular-weight thiol GSH,
which is maintained in the cytosol predominantly in a reduced form
(24, 35). GSH is a ubiquitous tripeptide
(
-glutamylcysteinylglycine) that has proposed functions in many
cellular processes, including the detoxification of various
carcinogens, mutagens, and ROS (34, 44). Studies with the
model eukaryote Saccharomyces cerevisiae have led to a
better understanding of the role of this peptide in cellular metabolism
and have shown that GSH is an essential metabolite that is required as
a reductant during normal growth conditions (reviewed in reference
16). This essential function may be the removal of
toxic intermediates which accumulate during the course of normal cell
metabolism (5, 18). In addition, yeast strains lacking GSH,
or altered in their GSH redox state, are sensitive to conditions of
oxidative stress induced by hydroperoxides and the superoxide anion, as
well as to the toxic products of lipid peroxidation (14, 15, 17,
19, 27, 48). This sensitivity to ROS presumably reflects the
antioxidant capacity of GSH both as a free-radical scavenger and as an
electron donor for glutathione peroxidases and transferases (16,
34).
In this present study, we demonstrate that protein S-thiolation occurs
in yeast and examine the role of GSH in the process. GAPDH is
identified as the most abundant S-thiolated protein during conditions
of oxidative stress, but, surprisingly, only the Tdh3 and not the Tdh2
GAPDH isoenzyme was modified. We present evidence that both enzymes are
required for survival during conditions of oxidative stress but play
complementary roles depending on their ability to undergo
S-thiolation.
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MATERIALS AND METHODS |
Yeast strains and media.
The S. cerevisiae
strains used in this study are described in Table
1. The strains were grown in rich YEPD
medium (2% [wt/vol] glucose, 2% [wt/vol] Bacto Peptone, 1%
[wt/vol] yeast extract) or minimal SD medium (0.17% [wt/vol] yeast
nitrogen base without amino acids, 5% [wt/vol] ammonium sulfate, 2%
[wt/vol] glucose) supplemented with appropriate amino acids and
bases: 2 mM leucine, 4 mM isoleucine, 1 mM valine, 0.3 mM histidine,
0.4 mM tryptophan, 0.15 mM adenine, and 0.2 mM uracil. The media were
solidified by the addition of 2% (wt/vol) agar.
Sensitivity to oxidants.
Dose-response curves were generated
by growing cells to the exponential phase (1 × 107 to
2 × 107 cells/ml) in SD medium at 30°C and treating
them with 4 mM H2O2 for 1 h. Aliquots of
cells were diluted into fresh YEPD medium at 20-min intervals and
plated in triplicate on YEPD plates to obtain viable counts after 3 days of growth. Sensitivity to H2O2, tert-butyl hydroperoxide, and diamide was determined by
spotting strains onto YEPD plates containing various concentrations of oxidants (18). Cells were grown to stationary phase in YEPD, and 10-µl aliquots of each culture diluted to an absorbance at 600 nm
of 2.0 were spotted onto appropriate plates. Sensitivity was determined
by comparison of growth to that of the wild-type strain after 3 days.
Analysis of protein S-thiolation.
The analysis of
S-thiolation in yeast was adapted from methods described for mammalian
cells (43, 46). Yeast cells were grown to exponential phase
(1 × 107 to 2 × 107 cells/ml) in
minimal SD medium and treated with 50 µg of cycloheximide per ml for
15 min to completely inhibit cytoplasmic protein synthesis (9). The cells were incubated with a final concentration of 0.51 nM L-[35S]cysteine for 1 h to
facilitate labelling of the intracellular pool of low-molecular-weight
sulfhydryls. They were then washed with SD medium and resuspended in SD
medium supplemented with the necessary auxotrophic requirements, as
well as H2O2 where desired. Cell extracts were
prepared in 20 mM sodium phosphate buffer (pH 7.4) containing 100 mM
phenylmethylsulfnonyl fluoride by breaking cells with glass beads,
using a Minibead Beater (Biospec Scientific), for 30 s at 4°C.
Parallel extracts were prepared in buffer containing either 50 mM
N-ethylmaleimide (NEM) to prevent thiolation during the
sample preparation or 25 mM dithiothreitol (DTT) to reduce any
S-thiolated proteins. To quantify protein S-thiolation, aliquots of
cell extracts were precipitated on Whatman GF/C glass microfiber
filters with 10% trichloroacetic acid. Radioactive incorporation was
measured by scintillation counting, and S-thiolation is expressed as
the difference between the NEM- and DTT-treated extracts (cpm per
microgram of protein).
Autoradiography, following sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE), was used to investigate the range of
proteins that were S-thiolated. Cell extracts (10 to 50 µg), diluted
in Laemmli buffer, were boiled (3 min), adjusted to 40 mM
iodoacetamide, and electrophoresed on 4 to 15% polyacrylamide SDS-PAGE
minigels (Bio-Rad). For autoradiography, the gels were stained in
0.125% (wt/vol) Coomassie blue-30% (vol/vol) methanol-10% (vol/vol) acetic acid, pretreated with En3Hance (NEN-Life
Sciences) as specified by the following manufacturer, and dried before
being exposed to film (Hyperfilm N; Amersham-Pharmacia).
HPLC and electrochemical detection of low-molecular-weight
sulfhydryls.
The preparation of yeast cell extracts for analysis
of low-molecular-weight sulfhydryls was based on that described for
tissue extracts (20). Cell pellets were broken in ice-cold
0.6 N perchloric acid containing 2 mM EDTA by vortexing with glass
beads. Following cell breakage, proteins were precipitated by
incubating on ice for 15 min and collected by centrifugation in a
microcentrifuge for 15 min at 4°C. The resulting supernatants were
diluted in a mobile phase before being separated by reverse-phase
chromatography on a C18 column (5 µm; 4.5 by 250 mm;
Shperisorb octadecyl silane 2) under isocratic conditions with 0.1 M
KH2PO4-0.35% (vol/vol) acetonitrile
(13) at a flow rate of 0.9 ml/min. To identify the range of
low-molecular-weight thiols involved in protein S-thiolation, the
protein precipitates were treated with 25 mM DTT at 37°C for 20 min
and the resulting supernatants were analyzed by high-performance liquid
chromatography (HPLC). Compounds were identified based on known
sulfhydryl standards including GSH, -Glu-Cys, and cysteine, which
were measured by electrochemical detection with a Coulochem II (ESA)
detector (E = + 550 mV).
GAPDH enzyme assays.
GAPDH activity was measured by the
method of McAlister and Holland (32) and is expressed as
micromoles of NADH formed per minute per microgram of protein.
Western blot analysis.
Protein extracts (20 µg/lane) were
electrophoresed under reducing conditions on 4 to 15%
polyacrylamide gradient SDS-PAGE minigels and electroblotted onto a
polyvinylidene difluoride membrane (NEN Research Products, Boston,
Mass.). The blot was incubated in 2 µg of anti-GAPDH monoclonal
antibody (MAB374; Chemicon International Inc., Temecula, Calif.) per
ml. Bound antibody was visualized by chemiluminescence (Renaissance;
NEN Research Products) following incubation of the blot in rabbit
anti-mouse immunoglobulin-horseradish peroxidase conjugate (1/2,000
dilution; DakoPatts, Carpinteria, Calif.).
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RESULTS |
Characterization of protein S-thiolation in yeast.
We first
established that the intracellular pool of sulfhydryls in yeast could
be radiolabelled by incubation with
L-[35S]cysteine as described in
Materials and Methods. Low-molecular-weight sulfhydryls were separated
by HPLC, and radioactivity was analyzed to determine the fate of
the radiolabel (Fig. 1A). The major peak comigrated with GSH, accounting for greater than 75% of the
radioactivity detected. In addition, two smaller peaks that
comigrated with cysteine and -glutamylcysteine were detected.
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FIG. 1.
HPLC separation of low-molecular-weight sulfhydryls. (A)
Sulfhydryls were separated by HPLC (see Materials and Methods)
following radiolabelling of the intracellular pool with
[35S]cysteine. The retention times of the known standards
cysteine (Cys), cysteinylglycine (C-G), -glutamylcysteine (-G-C),
and glutathione (GSH) are shown. (B) Sulfhydryls bound to proteins by
S-thiolation were released by treatment with DTT and separated by
HPLC.
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To characterize the effect of oxidative stress on protein S-thiolation,
a dose-response curve to H
2O
2 was generated by
challenging
labeled cells with H
2O
2 at
concentrations between 0.5 and 4 mM
for 30 min (Fig.
2A). Cell extracts were prepared in the
presence
of either NEM to prevent S-thiolation during the extraction
procedure
or DTT to reduce any S-thiolated proteins, and the level of
S-thiolation
was quantified as the difference between the NEM- and
DTT-prepared
extracts. The basal level of S-thiolation was very low in
unstressed
cells but increased at all concentrations of
H
2O
2 tested (Fig.
2A). The maximum level of
S-thiolation was observed at 2 mM H
2O
2,
which
was chosen for all subsequent thiolation experiments. To
determine the
number and range of proteins that are S-thiolated,
total-cell extracts
were examined by SDS-PAGE and autoradiography.
As expected from the
radioactive counts, few S-thiolated proteins
were detected in
unstressed cells (Fig.
2B) whereas several proteins
(e.g., of 45, 70, and 120 kDa) were detected following treatments
with
H
2O
2, including a prominent protein at
approximately 38 kDa.
Radioactivity incorporation was confirmed to
occur as a result
of S-thiolation since it was reversed by treatment
with the reducing
agent DTT (Fig.
2B, lanes +DTT).
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FIG. 2.
Protein S-thiolation following treatment with hydrogen
peroxide. Following radiolabelling of the intracellular pool of
low-molecular-weight sulfhydryls, cells were treated with various
concentrations of H2O2 for 30 min. (A) Protein
S-thiolation increased at all concentrations of
H2O2 tested. (B) Proteins were separated by
SDS-PAGE and analyzed by autoradiography. The cell extracts challenged
with 1, 2, and 4 mM H2O2 were treated with 25 mM DTT to reduce S-thiolated proteins (lanes +DTT). Molecular mass
markers are indicated in kilodaltons.
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To determine the role of GSH in protein S-thiolation, we examined yeast
strains in which
GSH1, encoding
-glutamylcysteine
synthetase,
GSH2, encoding glutathione synthetase, or
GLR1, encoding
glutathione reductase, was deleted. The
gsh1 mutant is completely
devoid of GSH, whereas, the
gsh2 mutant lacks GSH but can still
make the dipeptide
-glutamylcysteine (
-Glu-Cys) intermediate
in GSH synthesis
(
18). The
glr1 mutant contains a lowered
GSH
redox ratio (GSH-to-GSSG ratio) due to the inability to recycle
oxidized GSSG to the reduced GSH form (
15). A different
pattern
of protein S-thiolation was observed in the
gsh1
mutant compared
to the wild-type strain, with several novel proteins
labelled
during both stressed and nonstressed conditions (Fig.
3). In contrast,
the pattern of protein
S-thiolation in the
glr1 and
gsh2 mutants
was
similar to that in the wild-type strain. The role of GSH was
further
analyzed by determining the range of low-molecular-weight
sulfhydryls
that could participate in protein S-thiolation. The
proteins were
treated with the reducing agent DTT following the
S-thiolation
reaction, and released thiols were examined by HPLC
analysis
(Fig.
1B). A major peak corresponding to GSH was detected,
indicating
that GSH is the main thiol compound that undergoes
S-thiolation.
In addition, smaller peaks corresponding to cysteine
and
-Glu-Cys
were detected. Having established the range and
extent of protein
S-thiolation, we turned our attention to the
identification of the most
prominent, 38-kDa, protein detected.
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FIG. 3.
Protein S-thiolation in mutants affected in GSH
metabolism. Protein S-thiolation was analyzed following treatment with
2 mM H2O2 for 30 min in the wild-type (wt)
strain (CY4) and strains with GSH1 (-glutamylcysteine
synthetase), GSH2 (glutathione synthetase), and
GLR1 (glutathione reductase) deleted.
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Identification of GAPDH as the major S-thiolated protein in
yeast.
Given the size of the 38-kDa protein and the fact that
GAPDH has been identified as a target of S-thiolation in human
endothelial cells and blood monocytes (39, 42), we
investigated whether the 38-kDa yeast protein is GAPDH. Three unlinked
genes (TDH1, TDH2, and TDH3) encode
isoenzymes of GAPDH in yeast (33). Tdh2 and Tdh3 are highly
homologous (98% similarity, 96% identity), whereas Tdh1 is somewhat
less conserved (94% similarity and 88% identity to Tdh3). Strains
lacking both TDH2 and TDH3 are inviable, indicating that at least one of these genes must be present for normal
cell growth (33). Therefore, we examined protein
S-thiolation in mutants with TDH1, TDH1 and
TDH2, or TDH1 and TDH3 deleted. The 38-kDa S-thiolated protein was detected in strains that
contain Tdh3 (tdh1 tdh2) but not in strains that contain
Tdh2 (tdh1 tdh3), indicating that Tdh3 is the 38-kDa
S-thiolated protein (Fig. 4A). We
confirmed the identity of the 38-kDa protein as GAPDH by
immunoprecipitation with an anti-GAPDH antibody (data not shown). This
result is surprising given the high degree of sequence homology between
Tdh2 and Tdh3. In addition to the overall sequence conservation, the
yeast GAPDH proteins contain two active-site cysteine residues in
a conserved pentapeptide (C-T-T-N-C), which could serve as possible
sites for S-thiolation following an oxidative stress. This difference in protein S-thiolation could not be accounted for by differences in the relative levels of the Tdh2 and Tdh3 polypeptides (Fig. 4B)
(32). In addition, the Western blot analysis in Fig. 4B demonstrates that treatment with cycloheximide or
H2O2 did not affect the levels of the various
Tdh polypeptides. To determine whether S-thiolation of Tdh3 but not
Tdh2 is physiologically relevant, we next examined the effects of
oxidative stress on mutants lacking GAPDH isoenzymes.
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FIG. 4.
Protein S-thiolation in tdh mutants. (A)
Protein S-thiolation was analyzed following treatment with 2 mM
H2O2 for 30 min in the wild-type (wt) strain
(6B) and strains with TDH1, TDH1 and TDH2, or
TDH1 and TDH3, all encoding GAPDH, deleted. (B)
Western blot analysis probed with anti-GAPDH antibody.
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Sensitivity of tdh mutants to oxidants.
If
S-thiolation of Tdh3 serves to protect the GAPDH enzyme from
irreversible oxidation, we reasoned that strains lacking
TDH3 should be sensitive to oxidants. This was tested by
treating the various mutant strains with 4 mM
H2O2 for 1 h while monitoring cell
viability (Fig. 5). As predicted, strains
lacking TDH3 (tdh3 and tdh1 tdh3
strains) were extremely sensitive to H2O2
compared to the wild-type controls. The strain lacking both
TDH1 and TDH3 was the most sensitive, with no
survivors detected after 40 min of treatment. In this experiment, the
H2O2 stress was relieved by diluting the cells
into YEPD medium before plating to determine viable counts. We next
examined the effect of a constant exposure to oxidants by growing the
strains to stationary phase and spotting them onto plates containing
concentrations of H2O2 (4.5 mM) or tert-butyl hydroperoxide (2.5 mM) that allowed the wild-type
control to grow (Fig. 6). We reasoned
that strains containing Tdh3 as the major GAPDH enzyme (tdh2
and tdh1 tdh2 strains) would be unable to grow if
S-thiolation inhibits the activity of Tdh3. In agreement with this
prediction, the growth of the tdh1 and tdh3
mutants (contain Tdh2) was unaffected in the presence of hydroperoxides whereas the tdh2 and tdh1 tdh2 mutants did not
grow. Similarly, the tdh1 tdh3 double mutant was able to
grow in the presence of tert-butyl hydroperoxide but, in
contrast, was unable to grow in the presence of
H2O2, indicating that Tdh2 may also play some role during continued exposure to H2O2 stress.
In addition, none of the tdh mutants were sensitive to the
thiol oxidant diamide (Fig. 6), in agreement with the finding that it
does not promote protein S-thiolation (data not shown). However, all of
the tdh mutants were more resistant to diamide than were the
wild-type control, similar to the findings that mutants altered in
sulfhydryl regulation, including the GSH, glutaredoxin, and thioredoxin
systems, are also resistant to this thiol oxidant (18, 31,
36). We next examined the effect of H2O2
stress on GAPDH enzyme activity.
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FIG. 5.
Dose-response curves of tdh mutants to
hydrogen peroxide. The wild-type (wt) strains 6B and 29A and
strains with TDH1, TDH2, TDH3,
TDH1 and TDH2, or TDH1 and
TDH3 deleted were grown to exponential phase in SD
medium and treated with 4 mM H2O2 for 1 h.
The cells were diluted and plated in triplicate onto YEPD medium to
monitor cell viability at 20-min intervals. Percent survival is
expressed relative to the untreated control cultures (100%).
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FIG. 6.
Sensitivity of tdh mutants to a continued
exposure to oxidants. Sensitivity to H2O2 (4.5 mM), tert-butyl hydroperoxide (t-BH) (2.5 mM),
and diamide (3 mM) was determined by spotting strains onto YEPD plates
containing the appropriate oxidants. Cultures of the wild type (wt)
(6B) and mutants with TDH1, TDH2,
TDH3, TDH1 and TDH2, or
TDH1 and TDH3 deleted were grown to stationary
phase in YEPD and diluted to an absorbance at 600 nm of 2.0, and
10-µl aliquots were spotted onto appropriate plates. The plates were
incubated at 30°C for 2 days before growth was scored.
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Inhibition and recovery of GAPDH enzyme activity following exposure
to H2O2 stress.
The tdh1 tdh2,
tdh1 tdh3, and wild-type strains were grown to exponential
phase and treated with 2 mM H2O2 for 30 min
before GAPDH activity was determined (Fig.
7). Prior to the peroxide treatment, the
tdh1 tdh2 and tdh1 tdh3 mutants were found to
contain approximately 44 and 19% of the GAPDH activity found in the
wild-type strain. Following treatment with
H2O2, GAPDH activity was inhibited to
approximately 6 to 13% of its initial activity in all three strains
tested. This indicates that both the Tdh2 and Tdh3 GAPDH isoenzymes are
extremely sensitive to inactivation by oxidation. However, this may
either arise due to irreversible oxidation or due to S-thiolation of
the enzyme active site, both of which would inhibit GAPDH activity. To
distinguish between these possibilities, cells were transferred into
fresh SD medium to monitor the recovery of GAPDH activity once the
H2O2 stress was relieved. The tdh1 tdh2 mutant, which contains the S-thiolatible Tdh3 isoenzyme, recovered GAPDH activity to 55 and 95% of the starting activity following a 1- and 2-h growth period, respectively. In contrast, the
tdh1 tdh3 mutant recovered only 45% activity after 2 h. The wild-type strain contained 56% activity following the 2-h
recovery period, presumably reflecting the presence of both the Tdh2
and Tdh3 polypeptides.
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FIG. 7.
Inhibition and recovery of GAPDH enzyme activity
following exposure to H2O2 stress. GAPDH enzyme
activity (µmoles per minute per microgram) was determined in the
wild-type strain (6B) and in tdh1 tdh2 and tdh1
tdh3 mutants grown to exponential phase in SD medium (t = 0).
Cells were treated with 2 mM H2O2 for 30 min,
and aliquots were taken to determine GAPDH activity
(H2O2). The cells were washed and resuspended
in fresh SD medium to monitor the recovery of GAPDH activity for 60 or
120 min.
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DISCUSSION |
Hydrogen peroxide is a ubiquitous molecule that is formed as a
product of many oxidase-mediated reactions and of auto-oxidation of
hemoproteins and flavoproteins (38). As well as being both freely diffusible and reactive itself, H2O2
must be removed from cells to avoid formation of the highly reactive
hydroxyl radical. Detoxification can be mediated by catalases, which
catalyze its breakdown into H2O and O2, and by
glutathione peroxidases, which utilize GSH as a reductant. In yeast,
GSH-mediated reactions appear to be the primary means of detoxifying
this ROS, although catalases appear to provide an overlapping defense
system during particular growth conditions (16, 26). In
addition to the oxidant sensitivity of gsh1 and
glr1 mutants (15, 17),
H2O2 was found to increase the levels of
oxidized (GSSG), protein-bound (GSSP), and extracellular GSH at the
expense of intracellular GSH (16a). In the present study, we
have shown that the levels of GSSP are increased due to modification of
specific target proteins and that Tdh3 is the major S-thiolated protein.
Most of the S-thiolated proteins detected were formed due to reaction
with GSH, since this was the most abundant sulfhydryl released by
reaction with DTT, and the gsh1 mutant, which is
totally devoid of GSH (17), contained an altered
pattern of S-thiolation. Interestingly, the dipeptide -Glu-Cys may
be able to replace GSH in the S-thiolation reaction, since the pattern
observed in the gsh2 mutant was similar to that seen in the
wild type. We have previously shown that the gsh2 mutant is
devoid of GSH but contains elevated levels of the dipeptide
-Glu-Gys, which can substitute for GSH as an antioxidant in yeast
(18). The fact that the wild-type pattern of S-thiolation
was found in the glr1 mutant, which contains elevated levels
of GSSG (15, 19), argues against a mechanism that
proceeds via a thiol-disulfide exchange reaction with the
oxidized disulfide form of GSH (GSSG). Similarly, the 2 mM
H2O2 treatment used for these experiments did
not cause any increase in the level of GSSG (data not shown).
GAPDH was identified as the most abundant S-thiolated protein in yeast
following a challenge with H2O2. Previous
studies with mammalian cells have also identified GAPDH as a target of
S-thiolation, but the studies presented here show for the first time
that this is a regulated process and that it affects survival during
conditions of oxidative stress. GAPDH is a glycolytic enzyme that is
active as a tetramer of identical 37-kDa subunits catalyzing the
conversion of glyceraldehyde-3-phosphate to 1,3-diphosphoglycerate. It
has been extensively characterized in S. cerevisiae, in
which it is one of the most abundant soluble proteins and is encoded by
three unlinked genes designated TDH1, TDH2, and
TDH3 (1, 22). McAlister and Holland reported the
isolation of yeast mutants lacking combinations of the GAPDH isoenzymes
(33). None of the TDH genes were individually essential for cell viability, but a functional copy of either TDH2 or TDH3 was required since tdh2
tdh3 double mutants were inviable. These results implied that Tdh1
may perform a function distinct from that of Tdh2 or Tdh3, since Tdh1
alone was unable to support growth. Both the Tdh2 and Tdh3 polypeptides
are able to form catalytically active homotetramers, but the apparent
Vmax of the Tdh3 homotetramer is approximately
two- to threefold lower than that of the Tdh2 homotetramer
(32). Interestingly, two forms of the Tdh3 polypeptide that
differ in their isoelectric charge were detected by two-dimensional
PAGE and may represent catalytically different forms of the GAPDH
tetramer (32). The two forms of Tdh3 polypeptide did not
arise as a result of differential phosphorylation, and it is tempting
to speculate that they arose due to differences in protein
S-thiolation. Therefore, the relatively low Vmax
of Tdh3 may be due to the S-thiolated and hence inactivated form of Tdh3.
Given that the Tdh2 and Tdh3 polypeptides have 98% homology and are
functionally redundant for glycolysis, it is surprising that they
differ in protein S-thiolation. Both Tdh2 and Tdh3 GAPDH enzyme
activities were inactivated by exposure to
H2O2. However, Tdh3 (thiolated) activity was
recovered within 2 h after the removal of the
H2O2 stress, suggesting that the oxidative
inactivation was reversible. In contrast, Tdh2 (nonthiolated) activity
was restored to only 45% of the initial activity during the 2-h
recovery period. The S-thiolation of Tdh3 appears to be physiologically relevant since strains lacking TDH3 were hypersensitive to a
lethal dose of H2O2 compared to the wild type
and tdh2 strains. Thus, S-thiolation, and hence protection
of Tdh3 against irreversible oxidation, was required for survival
following a challenge with H2O2. However, the
nonthiolated Tdh2 polypeptide is also necessary during exposure to
oxidative-stress conditions, since it was required for growth during
the continued presence of oxidants. Our model to explain these results
postulates that during a prolonged exposure to low levels of oxidants,
the continued S-thiolation of Tdh3 would negatively affect cell growth,
but during such conditions, enough of the Tdh2 enzyme may avoid
oxidation to provide the necessary GAPDH activity for growth. At higher
concentrations of oxidants, which would irreversibly oxidize the Tdh2
polypeptide, the Tdh3 polypeptide could be protected by S-thiolation
until such time as the oxidative pressure is removed (Fig.
8).
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|
FIG. 8.
Model for the oxidant sensitivity of tdh
mutants. During continuous or low exposure to oxidants, Tdh3 is
S-thiolated (-SG), inhibiting GAPDH activity. Under these conditions,
Tdh2 must provide enough GAPDH activity for growth. Following an
exposure to high concentrations of oxidant, Tdh2 is irreversibly
oxidized (*). Tdh3 is protected by protein S-thiolation until such time
as the oxidant pressure is removed and the cells can resume growth.
|
|
In mammalian cells, the glycolytic synthesis of ATP is inactivated by
conditions of oxidative stress, mainly at the level of GAPDH, due to
three independent effects (7, 25): (i) direct inactivation
of the enzyme active site, (ii) a decrease in the cofactor NAD, and
(iii) a shift in the cytosolic pH from the enzyme optima (7,
25). Thus, protection of the GAPDH active site by S-thiolation
would protect against oxidative inactivation of this crucial glycolytic
enzyme. In addition, it has been proposed that blocking glycolysis at
the GAPDH step would be beneficial during conditions of oxidative
stress since it would result in an increased flux of glucose
equivalents through the pentose phosphate pathway, leading to the
generation of NADPH (39, 42). Such NADPH could provide
reducing power for antioxidant enzymes including catalases and the
GSH-GSH peroxidase system (21, 30). Evidence is also
accumulating that GAPDH functions in processes unrelated to glycolysis,
including, DNA repair and replication, translational control of gene
expression, and endocytosis and in a plasma membrane oxidoreductase
complex (reviewed in references 2 and
45). It is unknown whether S-thiolation of GAPDH
affects its activity in these various processes.
In addition to its role in protecting individual protein -SH groups
from oxidation, and given the high concentrations (millimolar) of
sulfhydryl groups in the cell, thiolation has been suggested to serve
as a general antioxidant defense system analogous to free-radical
scavengers (47). However, this seems unlikely from the
present data, since the tdh3 mutant, lacking the most
abundant S-thiolated protein, did not lead to increases in the level of thiolation of existing proteins or the appearance of new S-thiolated proteins. Rather, the consistent pattern of labelling indicates that
S-thiolation is a highly controlled form of posttranslational modification resulting from an oxidant challenge. This modification may
serve to protect the cysteine residues of particular proteins from
oxidation or, alternatively, may provide a novel form of protein
regulation. Two recent reports have indicated a role for reversible
thiolation in the regulation of enzyme activity. First, S-thiolation
was implicated in the regulation of the HIV-1 protease under the
control of glutaredoxin (11); second, S-thiolation was shown
to regulate the E1 and E2 ubiquitin-conjugating enzymes in bovine
retina cells (28). Here, we have demonstrated that the
activity of GAPDH in protection against oxidative stress is regulated
by protein S-thiolation and that this process is physiologically important for survival during conditions of oxidative stress.
| |
ACKNOWLEDGMENTS |
We thank Michael Holland for the GAPDH mutants and Daniel
Gonzalbo for the anti-GAPDH antibody.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: School of
Biochemistry & Molecular Genetics, University of New South Wales,
Sydney, NSW 2052, Australia. Phone: 61 (2) 9385 2031. Fax: 61 (2) 9385 1050. E-mail: c.grant{at}unsw.edu.au.
| |
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Abstract
Introduction
