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J. Biol. Chem., Vol. 276, Issue 26, 24261-24267, June 29, 2001
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From the Department of Cell Biology, Harvard Medical School,
Boston, Massachusetts 02115
Received for publication, February 16, 2001, and in revised form, April 2, 2001
The disaccharide trehalose, which accumulates
dramatically during heat shock and stationary phase in many organisms,
enhances thermotolerance and reduces aggregation of denatured
proteins. Here we report a new role for trehalose in protecting
cells against oxygen radicals. Exposure of Saccharomyces
cerevisiae to a mild heat shock (38 °C) or to a proteasome
inhibitor (MG132) induced trehalose accumulation and markedly increased
the viability of the cells upon exposure to a free
radical-generating system (H2O2/iron). When cells were returned to normal growth temperature (28 °C) or
MG132 was removed from the medium, the trehalose content and resistance
to oxygen radicals decreased rapidly. Furthermore, a mutant unable to
synthesize trehalose was much more sensitive to killing by oxygen
radicals than wild-type cells. Providing trehalose exogenously enhanced
the resistance of mutant cells to H2O2.
Exposure of cells to H2O2 caused oxidative
damage to amino acids in cellular proteins, and trehalose accumulation
was found to reduce such damage. After even brief exposure to
H2O2, the trehalose-deficient mutant exhibited
a much higher content of oxidatively damaged proteins than wild-type
cells. Trehalose accumulation decreased the initial appearance of
damaged proteins, presumably by acting as a free radical scavenger.
Therefore, trehalose accumulation in stressed cells plays a major role
in protecting cellular constituents from oxidative damage.
Reactive oxygen species
(ROS),1 especially hydrogen
peroxide (H2O2), superoxide anions, and
hydroxyl radicals, are generated in cells through normal metabolic
activity, by ionizing or ultraviolet radiation, and by macrophages and
neutrophils during phagocytosis (1). These ROS can damage proteins by
causing modifications of amino acid side chains, formation of
cross-links between proteins, and fragmentation of the polypeptide
backbone (2). In addition, ROS can modify bases and sugars in DNA,
leading to DNA chain breaks (3), and cause lipid peroxidation in cell
membranes (4). Oxidative damage to cellular components may contribute
to the aging process and plays an important role in the pathogenesis of
inflammatory diseases and reperfusion injury (1, 5). Moreover, in yeast
and bacteria, oxidative damage also contributes to the cell death
occurring upon exposure to high temperatures or at stationary phase (6,
7). A number of biochemical systems have evolved to protect cells
against ROS, including enzymes (e.g. catalase and superoxide
dismutase) as well as the nonenzymatic protective molecules,
glutathione and thioredoxin (5, 8).
Exposure of cells to elevated temperatures or to other harsh treatments
that damage cell proteins (e.g. hydrogen peroxide or
ethanol) induces the heat shock response (9). This response increases
the ability of the cells to resist a subsequent exposure to a high and
otherwise lethal temperature or toxic agents. This induced tolerance to
harsh conditions is generally believed to be caused by the synthesis of
heat shock proteins (HSPs) (10). Many HSPs are molecular chaperones
that promote the refolding of damaged proteins (11, 12). Other HSPs are
components of the proteolytic machinery of the cell, which degrade
irreversibly damaged proteins (e.g.
ATP-dependent proteases in bacteria and ubiquitin in
eukaryotic cells) (13, 14).
In bacteria and yeast, the nonreducing disaccharide trehalose
( When yeast or mammalian cells are exposed to a mild heat shock or to
low concentrations of hydrogen peroxide, they also develop increased
resistance to lethal doses of hydrogen peroxide (26-29). Catalase and
superoxide dismutase, the synthesis of which increases during heat
shock or exposure to oxidants (26, 30-32), contribute to this enhanced
resistance. However, the cellular mechanisms that actually provide
protection against ROS are not well defined. Although the possible
involvement of trehalose in protection against oxygen radicals has
never been studied, two findings led us to hypothesize that it may
serve such a role: (i) large amounts of trehalose accumulate in yeast
during exposure to oxidants (16), and (ii) mannitol, a sugar alcohol,
is known to be a potent free-radical scavenger (33, 34). The present
studies were undertaken to investigate whether trehalose may play an
important role in protecting yeast against oxidative damage. We have
tested whether various treatments that stimulate trehalose production
or that reduce trehalose content cause concomitant changes in the
resistance of the cell to oxygen radicals. In addition, we used a yeast
mutant that cannot synthesize trehalose to investigate whether this
sugar is in fact responsible for the changes in resistance to oxidants and whether this disaccharide may also reduce oxidative damage to cell proteins.
Saccharomyces cerevisiae Strains--
The ise1
strain JN284: MAT Assay of Cell Resistance to
H2O2--
Cells were grown at 25 or 28 °C
until mid-log phase and then either subjected to a mild heat shock (35 or 38 °C) or exposed at 28 °C to 50 µM proteasome
inhibitor MG132 (in 0.1% Me2SO) (kindly provided by
Proscript, Inc.) for varying periods to enhance trehalose accumulation.
After these pre-treatments, cells were incubated with 2 mM
H2O2 and 1 mM FeCl3 at
28 °C for the indicated times. In preliminary experiments, similar
toxic effects were obtained with H2O2 alone,
H2O2/FeSO4, or
H2O2/FeCl3, but in these studies, FeCl3 was added to maintain a high level of
Fe3+ and Fe2+. Cells were then diluted at least
100-fold in yeast extract-peptone-dextrose medium or yeast
extract-peptone-galactose medium and plated in duplicate to determine
the number of viable colonies.
Assay of Trehalose--
Trehalose was extracted from yeast cells
and assayed as described previously (22, 36). Briefly, trehalose was
extracted by boiling the cell pellet at 95 °C for 20 min. After
centrifugation, the level of trehalose was assayed in the supernatant
by conversion to glucose with trehalase (Sigma), which was
measured by a glucose assay kit (Sigma). The pre-existing glucose in
each sample was determined in a control reaction lacking trehalase and
subtracted from the total glucose.
Measurement of Carbonyl Groups in Proteins--
Cells were grown
until mid-log phase and then subjected to a mild heat shock (35 or
38 °C) for the indicated times. Cells were then incubated with
H2O2/FeCl3 as indicated, collected
by centrifugation, and lysed with glass beads. The carbonyl groups in
proteins were derivatized to 2,4-dinitrophenylhydrazones using the
OxyBlot detection kit (Intergen). Briefly, 10 µg of cellular proteins
were incubated with 2,4-dinitrophenylhydrazine for 15 min at room
temperature. Proteins were separated by 12% SDS-polyacrylamide gel
electrophoresis, and the derivatized carbonyl groups were detected with
a 2,4-dinitrophenyl-specific antibody and the ECL detection system
(Amersham Pharmacia Biotech). Coomassie Blue staining was used to
ensure that equal amounts of proteins were loaded onto the gel.
Cell Resistance to Oxygen Radicals Correlates with Trehalose
Content--
In yeast, trehalose is barely detectable during
logarithmic phase but accumulates to very high levels during heat shock
or under other stressful conditions (15-17). To investigate whether this accumulation of trehalose may enhance the resistance of the cells
to oxidants, exponentially growing wild-type yeast cells were exposed
to a mild heat shock at 38 °C. After different times, trehalose
content was determined, and the resistance of the cells to oxygen
radicals was measured by incubation with an oxygen radical-generating system, composed of H2O2 and FeCl3,
at 28 °C. H2O2 can react with Fe3+ to produce superoxide anions and Fe2+,
which can subsequently react with H2O2 to
generate hydroxyl radicals by the Fenton reaction (3, 4). These
conditions were chosen to cause rapid killing of most of the cells
within the first 15-20 min of exposure to the oxidants (data not
shown), i.e. before cells might induce antioxidant defense
mechanisms. After exposure to H2O2, the
cultures were plated to determine the fraction of cells that remained
viable. In the cells maintained at 28 °C, trehalose was not
detectable, and cell resistance to oxidants did not change
significantly. However, when the cells were incubated at 38 °C,
trehalose content increased dramatically, as did resistance to
H2O2 (Fig.
1A).
After a mild heat shock, if cells are returned to 28 °C their
content of trehalose falls very rapidly (16, 17). To test whether the
decrease in trehalose content is associated with a decrease in
resistance to oxidant, we incubated cells at 38 °C for 30 min and
then returned them to 28 °C. At different times after the downshift
in temperature, trehalose content and resistance of the cells to oxygen
radicals were determined. After the heat-shocked cells were returned to
28 °C, trehalose content fell to a very low level by 20 min. The
resistance of the cells to oxygen radicals also decreased concomitantly
(Fig. 1B).
We previously showed that treatment of yeast with the proteasome
inhibitor MG132 induced trehalose accumulation and enhanced thermotolerance (22). Moreover, when the inhibitor was removed, the
trehalose level and thermotolerance decreased concomitantly. To see
whether the rapid changes in trehalose content induced by MG132 also
correlate with alterations in resistance to oxygen radicals, we
incubated ise1 cells (a strain permeable to proteasome inhibitors) with MG132 during exponential growth at 28 °C. After different times, the cells were exposed to
H2O2, and their resistance to oxygen radicals
was measured. Upon incubation with MG132, the trehalose level increases
together with the resistance of the cells to a subsequent exposure to
oxygen radicals (Fig. 2A).
We then tested whether the decrease in trehalose level after removal of
MG132 also correlates with a reduction in the resistance of the cells
to oxygen radicals. Therefore, we incubated growing ise1
cells for 2 h with MG132 and then washed the cells and resuspended them in medium lacking MG132. At different times, the washed cells were
exposed to H2O2, and the number of viable cells
was measured. After removal of MG132, the trehalose content decreased
dramatically to its basal level within 30 min. The resistance of the
cells to H2O2 decreased most markedly within 30 min to a level close to that of the control cells (Fig. 2B).
After falling rapidly, the cellular resistance to
H2O2 remained slightly higher than that of
untreated cells, presumably because of the generation of other
protective factors during the treatment with MG132. It is noteworthy
that MG132 treatment also causes production of HSPs; but after removal
of the proteasome inhibitor, the level of HSPs did not fall and
actually continued to rise, in contrast to that of trehalose (22).
Likewise, upon return to 28 °C after a heat shock, the level of
induced HSPs, unlike that of trehalose (Fig. 1B), stayed
high for several hours (37). Thus, cellular resistance to oxidants
correlates closely with the level of trehalose but not with the level
of HSPs.
A stricter comparison of the time course of changes in trehalose level
and oxidant sensitivity is probably impossible because trehalose
content changes rapidly and can only be measured at a single time
point, whereas killing by H2O2 is a slow
time-dependent process and requires exposure for at least
several minutes (during which the trehalose level changes).
Nevertheless, it is noteworthy that even with 10 min exposure to
peroxide (Fig. 1), a surprisingly tight correlation between trehalose
content and resistance to oxidants was found. Altogether, these
different experiments indicate that the level of trehalose closely
correlates with and therefore may determine the resistance of the cells
to oxidants.
Trehalose Is Essential for Cell Survival during Oxidative
Stress--
The above findings strongly suggest that the accumulation
of trehalose enhances cellular resistance to oxygen radicals. However, a mild heat shock and MG132 also induce the production of HSPs, which
include the antioxidant enzymes catalase and mitochondrial superoxide
dismutase (26, 27, 30). To determine whether trehalose is in fact
responsible for the enhanced protection against oxidative damage, we
analyzed the survival of a yeast mutant lacking the two enzymes
catalyzing the synthesis of trehalose, trehalose-6-phosphate synthase
(TPS1) and trehalose-6-phosphate phosphatase (TPS2) (38, 39). In this
double mutant, the accumulation of trehalose as well as biosynthetic
intermediates is prevented (35). One potential complication in using a
tps1 mutant is that at 39 °C, this mutant not only fails
to accumulate trehalose but also does not produce HSPs such as Hsp104
and catalase (40). However, Singer and Lindquist (23) reported that at
35 °C, the production of HSPs increases in the tps1
mutant as in the wild type. Therefore, we grew the wild-type and
tps1tps2 mutant at 25 °C until log phase and shifted them
to 35 °C to induce trehalose accumulation in the wild type, while
allowing HSP production both in the wild type and tps1tps2 mutant. After 30 min, the cells were incubated with
H2O2, and at different times the fraction of
viable cells was determined. As expected, the tps1tps2
mutant strain did not accumulate trehalose at 35 °C (Table
I), and when exposed to
H2O2 it displayed a much greater sensitivity
than did the wild type (Fig.
3A). For example, after 60 min
of incubation with H2O2, only 0.2% of mutant
cells survived, whereas 55% of the wild-type cells were viable. This 300-fold reduction in survival in the mutant must have been caused by
the absence of trehalose, because the mutant and wild type produced
equal amounts of HSPs, as shown by the equivalent accumulation of
Hsp104, which was monitored because its level dramatically increases
during heat shock (41) (Fig. 3B). In addition, the activity
of catalase, an HSP that destroys H2O2, was not
lower in the mutant (Table I). In fact, its catalase activity
consistently appeared higher than in the wild type, perhaps indicating
a compensatory increase in this antioxidant enzyme.
To investigate whether trehalose by itself can reduce the high
sensitivity of the tps1tps2 mutant to oxidants, we tested
whether the addition of trehalose into the medium at the high
concentrations observed in cells during heat shock (up to 500 mM) (18) increases cell resistance to
H2O2. When the tps1tps2 mutant was
incubated in a medium containing trehalose before the addition of
H2O2, the number of cells remaining viable was
much greater than in nonsupplemented medium. Indeed, the addition of
500 mM trehalose increased by 20-fold the number of cells
viable after a 60-min exposure to H2O2 (Fig.
4). The addition of equal concentrations of mannitol or galactose, both of which are known to scavenge oxygen
radicals (42, 43), also increased the resistance of cells to oxygen
radicals, although to a slightly lesser extent than trehalose (Fig. 4).
By contrast, the addition of sucrose, another nonreducing disaccharide,
did not enhance cell survival. Thus, the addition of trehalose and
certain carbohydrates (but not all) can partially correct the defect in
the mutant.
Trehalose Accumulation Reduces Oxidative Damage to Cellular
Proteins--
One possible mechanism by which trehalose may enhance
cell resistance to H2O2 would be by preventing
oxidant-induced damage to critical cellular components such as proteins
or DNA. We investigated whether the exposure of yeast to
H2O2 causes oxidative damage to proteins and
whether the trehalose accumulation upon a mild heat shock decreases
such damage. The reaction of proteins with oxygen radicals leads to the
appearance of carbonyl groups in polypeptide chains (2). The resulting
carbonyl-containing residues can react with 2,4-dinitrophenylhydrazine
to generate the phenylhydrazone derivative. It is therefore possible to
assay the appearance of oxidant-modified amino acids in proteins by
using a 2,4-dinitrophenyl-specific antibody (44). Exponentially growing
W303-1a cells were either maintained at 28 °C or shifted to
38 °C for 1 h to induce trehalose accumulation. These cultures
were then exposed to H2O2 at 28 °C, and
after different times, we measured the appearance of carbonyl groups in
cell proteins by immunoassay as described above. When the yeast were
exposed to H2O2, the amount of carbonyl groups in cell proteins increased with the duration of the exposure to the
oxygen radicals (Fig. 5). Although
multiple cell proteins contained carbonyl groups, certain protein bands
seemed to be selectively modified, perhaps because they are especially
sensitive to oxidative damage, as has been observed previously for
certain proteins in bacteria and yeast (45, 46). After a mild heat shock, the cells showed lower amounts of carbonyl groups in all these
bands than did the cells maintained at 28 °C (Fig. 5). Thus, a heat
shock, while enhancing cell resistance to H2O2,
also provides protection of cellular proteins from oxidants.
To test whether the heat shock-induced accumulation of trehalose might
be responsible for this protection, we compared the amount of
carbonyl-containing proteins generated in the wild type and the
tps1tps2 mutant upon exposure to
H2O2. Cells were shifted from 25 to 35 °C
for 30 min to induce trehalose accumulation and then exposed to
H2O2 for 30 min at 25 °C. In the mutant
unable to synthesize trehalose, the amount of carbonyl groups generated in proteins was much greater than in the wild type (Fig.
6A).
A dramatic reduction in protein oxidation could be, in principle,
caused by an ability of trehalose to protect proteins from oxygen free
radicals or perhaps to promote the selective degradation of the
oxidant-damaged proteins. However, the latter possibility seems quite
unlikely because a much greater carbonylation of cell proteins was also
observed in the trehalose-deficient cells after an exposure to
H2O2 of only 10 min (Fig. 6B). In
prior studies, carbonyl-containing proteins were found to be slowly
degraded in cells (e.g. 30% of breakdown after 24 h)
(47). To explain the present findings, degradation would have to be
nearly complete in 10 min, despite continuous oxidation of proteins by
H2O2. Therefore, trehalose accumulation must
inhibit the covalent modification of proteins by oxygen radicals, and
this effect seems to account in a large part for the protection
provided by heat shock.
Trehalose is found in organisms as diverse as plants, insects, and
invertebrates, as well as eukaryotic microorganisms and bacteria, in
which it accumulates dramatically during stationary phase, heat shock,
and oxidative stress (19). Trehalose was long believed to function as a
carbohydrate reserve; however, subsequent studies showed that this
sugar has an important role in protecting cells against thermal damage
(21-23, 48). We demonstrate here a new and distinct function for
trehalose in protecting cells against oxygen radicals. We have shown
that the ability of cells to withstand the lethal effect of oxygen
radicals rises with the increase in their trehalose content upon a mild
heat shock or exposure to a proteasome inhibitor. Furthermore, upon
termination of these stimuli, trehalose content decreases rapidly to
the low levels seen in untreated cells, and the resistance of the cells to the oxidants also falls. Concomitant changes in trehalose content and resistance to oxygen radicals were observed in two strains (W303-1a and ise1) with two different growth media and four
different experimental treatments. Although large changes in trehalose
level occurred within 10 min after the downshift in temperature, these correlations were observed for short (10 min) or longer (60 min) times
of exposure to H2O2 (Fig. 1 and data not
shown). Therefore, these findings actually indicate that the initial
trehalose content in the cell correlates with its resistance to a
subsequent exposure to oxidants. Moreover, by using a
trehalose-deficient mutant, we showed that trehalose accumulation is
responsible for this increased resistance to oxygen radicals.
Although heat shock and the proteasome inhibitor MG132 also stimulate
the expression of various HSPs, their production cannot account for the
alterations in sensitivity to oxidants shown here. In previous studies,
we demonstrated that when the inhibitor MG132 was removed, the level of
various HSPs did not decrease, whereas trehalose and thermotolerance
fell rapidly (22). Thermotolerance thus does not correlate with the
level of HSPs. In the present experiments, when MG132 was removed or
when heat-shocked cells were returned to 30 °C, resistance to oxygen
radicals also fell within 30 min to levels close to those of control
cells. Further evidence that the level of HSPs does not correlate with
sensitivity to H2O2 was the finding that the
trehalose-deficient mutant, although able to induce HSPs such as
catalase at 35 °C, still required trehalose for the associated
increase in resistance to oxidants. Presumably, trehalose functions
in vivo synergistically with the antioxidant enzymes
(catalase, superoxide dismutase, and peroxidases) to protect cells from
oxygen radicals.
Perhaps the strongest evidence for the protective role of
trehalose was that its addition to the medium markedly enhanced the
resistance of the cells to oxygen radicals. Most likely, trehalose enhances resistance to oxidative stress by quenching oxygen radicals generated either in the medium by the H2O2/iron
mixture or inside cells after entry of H2O2.
Enhanced survival was also observed when the sugar alcohol mannitol or
the monosaccharide galactose was added to the medium, although
trehalose seemed more potent in protecting cells. Both mannitol and
galactose have been shown to scavenge hydroxyl radicals in
vitro (42, 43). This protective effect, however, is not a general
property of all sugars because another disaccharide, sucrose
( A role for trehalose in protection against oxidative damage has not
been proposed previously in the literature. However, several findings
are consistent with such a role. Expression of the
trehalose-6-phosphate phosphatase (TPS2), which catalyzes the second
step in trehalose biosynthesis, requires the stress response
element-binding protein, YAP1, which controls the expression of
other antioxidant proteins including thioredoxin and catalase (49). In
addition, Godon et al. (32) found that TPS2 was among the
proteins induced during adaptation to H2O2 in yeast.
Defense against oxygen radicals also seems important when cells are
exposed to elevated temperatures or during stationary phase, two
conditions in which trehalose production rises dramatically. In fact,
oxygen radicals contribute to the loss of cell viability when yeast are
shifted to 50 °C, and both catalase and superoxide dismutase help
cells to resist lethal high temperatures (6). In addition, in bacteria,
oxidative damage to proteins is increased during stationary phase (7).
The particular sensitivity of the trehalose-deficient cells to heat
shock has been attributed to its role in preventing protein
aggregation. However, trehalose may also help cells to survive heat
shock by reducing oxidative damage at high temperatures. Similarly,
trehalose production may be important in pathogenic microorganisms as a
mechanism to enhance resistance to the oxidants generated by phagocytic
cells and therefore may contribute to their virulence. A similar role
has been proposed for mannitol in the fungus Cryptococcus
neoformans (50). Mannitol produced by this fungus was shown to
scavenge reactive oxygen species generated by human neutrophils during
respiratory burst (50). Similarly, when the fungus Alternaria
alternata infects a plant, it secretes mannitol presumably to
scavenge oxygen radicals produced by the host plant (50, 51).
Trehalose has not been found thus far in mammals, although early
studies have not examined cells during heat shock or oxidative stress.
It is possible that other polyols might have evolved in mammalian cells
to serve a similar protective role. In fact, inositol and sorbitol have
been found to accumulate in tissue culture during oxidative stress (52)
and osmotic stress (53, 54), and therefore it is tempting to speculate
that they might also be important in protecting against free radicals.
When the trehalose content in the cells is low (e.g. when
yeast grow at 28 °C or in the tps1tps2 mutant), cellular
proteins are more susceptible to covalent damage by free radicals, as
shown here by the increased appearance of carbonyl groups. This ability of trehalose to reduce covalent modifications of proteins by oxidants indicates a new property of this sugar that can not be simply explained
by its ability to prevent protein aggregation and to facilitate
refolding of damaged polypeptides by chaperones (23). Many proteins in
yeast showed such modifications, and trehalose accumulation seemed to
reduce oxidative damage to all these proteins to a similar extent, as
would be expected if trehalose functions by scavenging free radicals.
By rapidly reacting with ROS, trehalose would prevent their reaction
with proteins and other cellular constituents (e.g. DNA,
RNA, or lipids). Such an ability to soak up ROS has been demonstrated
for mannitol and glycerol, both of which in vitro can reduce
oxidative damage to proteins (33, 34, 55). The ability of trehalose to
reduce oxidant-induced modifications of proteins and its capacity to
prevent protein aggregation probably involve different chemical
mechanisms. However, because oxidantly damaged proteins tend to be
denatured and to aggregate, both mechanisms may be important in
protecting against oxidative stress.
Oxidatively damaged proteins tend to aggregate, presumably because the
oxidative modification of critical residues leads to protein unfolding
(56, 57), and these abnormal proteins are selectively degraded in cells
(55, 58). It is possible that trehalose accumulation may also promote
the degradation of these potentially toxic proteins (e.g. by
reducing oxidative damage to the proteolytic machinery of the cell or
by maintaining the damaged polypeptides in an easily degraded soluble
form). Such an effect, however, cannot account for the present
findings. The trehalose-deficient cells showed a much greater content
of oxidatively damaged proteins even after a very short exposure to
H2O2, which indicates that trehalose primarily
prevents the initial damage by free radicals rather than facilitating
long-term cellular responses such as the selective breakdown of
oxidatively damaged proteins. Certain essential proteins may be
especially susceptible to damage by oxygen radicals, and the capacity
of trehalose to prevent such damage probably contributes to its ability
to prevent cell death upon oxidative stress. The present findings also
strongly suggest that trehalose should also protect other essential
cellular constituents (e.g. DNA or lipids) from damage by
oxygen radicals.
We thank J. Thevelein and B. Futcher for
providing the yeast strains.
*
This work was supported by research grants from
NIGMS, National Institutes of Health and the Amyotrophic Lateral
Sclerosis Association.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Present address: Korean Research Institute of Bioscience and
Biotechnology, Taejon, Korea, 305-333.
¶
To whom correspondence should be addressed. Tel.:
617-432-1855; Fax: 617-232-0173; E-mail:
Alfred Goldberg@hms.harvard.edu.
Published, JBC Papers in Press, April 11, 2001, DOI 10.1074/jbc.M101487200
The abbreviations used are:
ROS, reactive
oxygen species;
HSP, heat shock proteins;
TPS1, trehalose-6-phosphate
synthase;
TPS2, trehalose-6-phosphate phosphatase.
Trehalose Accumulation during Cellular Stress Protects Cells and
Cellular Proteins from Damage by Oxygen Radicals*
,
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-glucopyranosyl-1,1--D-glucopyranoside)
also accumulates to high concentrations during heat shock and
stationary phase (15-17). In yeast, the concentration of trehalose can
be as high as 500 mM during heat shock (18), and the
accumulation of this disaccharide is important for the associated
thermotolerance (19). Inactivation of the enzymes involved in trehalose
biosynthesis reduces the level of thermotolerance induced by exposure
to a mild heat shock and at stationary phase (20, 21). In addition, we
found that treatment of yeast with proteasome inhibitors
(e.g. MG132) caused an accumulation of trehalose as well as
HSPs and increased thermotolerance (22). Upon removal of these
inhibitors, thermotolerance fell rapidly together with the trehalose
content, whereas the level of HSPs did not decrease. Thus,
thermotolerance correlated closely with the trehalose content in the
cell but not with the level of HSPs (22). Trehalose has been shown to protect proteins against thermal inactivation (18), and Singer and
Lindquist (23) demonstrated that this sugar reduces protein aggregation
and maintains polypeptide chains in a partially folded state, thus
facilitating their refolding by cellular chaperones. Although trehalose
has not been found thus far in mammals, this sugar has been found
to improve the tolerance of mammalian cells to dessication (24) and
cryopreservation (25).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
his3 leu2 ura3 ise1)
was provided kindly by J. C. Wang (Harvard University) and was
grown in SD medium (0.67% yeast nitrogen base, 2% glucose, and
amino acids). The tps1tps2 mutant, a
generous gift from J. Thevelein and S. Hohman (Katholieke Universiteit
Leuven, Belgium), is YSH6.127.-3C (MATa ade2 his3 leu2 trp1 ura3
can1-100 GAL SUC2 tps1
::TRP1
tps2::LEU2) and is of the genetic background of
W303-1a (35). W303-1a was grown in yeast
extract-peptone-dextrose or yeast extract-peptone-galactose medium, and tps1tps2 mutant was grown in yeast
extract-peptone-galactose medium.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (26K):
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Fig. 1.
Changes in trehalose content correlate with
changes in the resistance of cells to oxygen radicals.
A, wild-type cells (W303-1a) growing at 28 °C were
switched to 38 °C (closed circles) or maintained at
28 °C (open circles) for 30 min. At the
indicated times, aliquots of cells were taken either to measure
trehalose content (upper panel) or to determine the fraction
of viable cells after exposure to
H2O2/FeCl3 for 10 min at 28 °C
(lower panel). B, after a 30-min incubation at
38 °C (closed circles) or 28 °C (open
circles), W303-1a cells were returned to 28 °C. At the
indicated times, aliquots were taken either to measure trehalose
content (upper panel) or to determine the fraction of viable
cells after exposure to H2O2/FeCl3
for 10 min (lower panel). The fraction of viable cells was
determined by using cells incubated without
H2O2 as the total cell number. The data are
mean values ± S.D. from three independent experiments.
View larger version (31K):
[in a new window]
Fig. 2.
The proteasome inhibitor MG132 triggers
concomitant changes in trehalose accumulation and resistance of cells
to H2O2. A, exponentially
growing ise1 cells were incubated with 50 µM
MG132 in 0.1% Me2SO (closed circles) or with
0.1% Me2SO alone (open circles). At the
indicated times, cells were exposed to
H2O2/FeCl3 for 60 min, and the
fraction of viable cells was measured (lower panels).
B, after a 2-h incubation with MG132 (closed
circles) or 0.1% Me2SO (open circles), the
cells were washed and resuspended in medium with 0.1%
Me2SO. At the indicated times, cells were exposed to
H2O2/FeCl3 for 60 min, and the
number of surviving cells was determined (lower panel). The
fraction of viable cells was determined by using cells incubated
without H2O2 as the total cell number. The data
are means ± S.D. from three independent experiments. The data on
trehalose content upon exposure of MG132 (A, upper
panel) and after removal of MG132 (B, upper
panel) are from Lee and Goldberg (22).
Catalase activity and trehalose content in wild-type and trehalose
mutant cells
View larger version (35K):
[in a new window]
Fig. 3.
Resistance to H2O2
is reduced in cells unable to synthesize
trehalose. A, survival of the wild type (WT)
and tps1tps2 mutant in the presence of
H2O2. W303-1a (closed
circles) or tps1tps2 mutant (open
circles) cells were grown at 25 °C until mid-log phase and then
were transferred to 35 °C for 30 min. After this mild heat shock,
the cells were incubated with
H2O2/FeCl3 at 25 °C. At the
indicated times, the fraction of viable cells was determined. The data
are means ± S.D. from three independent experiments.
B, Hsp104 production in wild-type (WT) and
tps1tps2 mutant cells. W303-1a or tps1tps2
mutant cells were grown at 25 °C until mid-log phase and then were
transferred to 35 °C or maintained at 25 °C for 30 min. After the
mild heat shock, the cells were collected and lysed. The content of
Hsp104 in the cell extract (15 µg) was measured by immunoblotting
with an Hsp104-specific antibody (Affinity Bioreagents, Inc.) and
subsequent detection by ECL.
View larger version (19K):
[in a new window]
Fig. 4.
Trehalose addition to the medium enhances
cell resistance to H2O2.
Tps1tps2 mutant cells were grown at 25 °C until mid-log
phase. The cells were collected, resuspended in medium without
(none, open circles) or with 500 mM trehalose (Tre, closed circles),
galactose (Gal, closed triangles), mannitol
(Man, open squares), or sucrose (Suc,
closed triangles), and incubated at 35 °C for 30 min. The
cultures were then exposed to
H2O2/FeCl3 at 25 °C. At the
indicated times, the fraction of viable cells was determined. The data
are means from three independent experiments.
View larger version (59K):
[in a new window]
Fig. 5.
A mild heat shock reduces oxidative damage to
cellular proteins. Exponentially growing W303-1a cells were
transferred from 28 to 38 °C or maintained at 28 °C for 1 h.
Both cultures were then exposed to 2 mM
H2O2 and 1 mM FeCl3 at
28 °C. At the indicated times, aliquots of cells were lysed by glass
beads. The amount of carbonyl groups in proteins was measured as
described under "Experimental Procedures."
View larger version (58K):
[in a new window]
Fig. 6.
Oxidative damage to cellular proteins
increases in cells unable to accumulate trehalose. W303-1a or
tps1tps2 mutant cells were grown at 25 °C until mid-log
phase and then transferred to 35 °C for 30 min. After this mild heat
shock, cells were exposed to 2 mM
H2O2 and 1 mM FeCl3 for
30 min (A) or with 5 mM
H2O2 and 1 mM FeCl3 for
10 min (B) at 25 °C, and the amount of carbonyl groups in
cell proteins was determined. WT, wild type.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-glucopyranosyl-1,2-
-D-fructofuranoside), was not able to protect the cells from H2O2.
This difference in enhancing resistance to oxygen radicals is
surprising because both are nonreducing disaccharides. It remains to be
established whether this difference is caused by a lesser ability of
sucrose to quench oxygen radicals or to enter the cells.
ACKNOWLEDGEMENTS
FOOTNOTES
Supported by postdoctoral fellowships from Association pour la
Recherche contre le Cancer and Human Frontier Science Program Organization.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.
Stadtman, E. R.,
and Berlett, B. S.
(1997)
Chem. Res. Toxicol.
10,
485-494
2.
Berlett, B. S.,
and Stadtman, E. R.
(1997)
J. Biol. Chem.
272,
20313-20316
3.
Henle, E. S.,
and Linn, S.
(1997)
J. Biol. Chem.
272,
19095-19098
4.
Halliwell, B.,
and Gutteridge, J. M.
(1984)
Biochem. J.
219,
1-14
5.
Ames, B. N.,
Shigenaga, M. K.,
and Hagen, T. M.
(1993)
Proc. Natl. Acad. Sci. U. S. A.
90,
7915-7922
6.
Davidson, J. F.,
Whyte, B.,
Bissinger, P. H.,
and Schiestl, R. H.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
5116-5121
7.
Dukan, S.,
and Nystrom, T.
(1998)
Genes Dev.
12,
3431-3441
8.
Moradas-Ferreira, P.,
Costa, V.,
Piper, P.,
and Mager, W.
(1996)
Mol. Microbiol.
19,
651-658
9.
Lindquist, S.
(1986)
Annu. Rev. Biochem.
55,
1151-1191
10.
Lindquist, S.,
and Craig, E. A.
(1988)
Annu. Rev. Genet.
22,
631-677
11.
Georgopoulos, C.,
and Welch, W. J.
(1993)
Annu. Rev. Cell Biol.
9,
601-634
12.
Hartl, F. U.
(1996)
Nature
381,
571-579
13.
Parsell, D. A.,
and Lindquist, S.
(1993)
Annu. Rev. Genet.
27,
437-496
14.
Sherman, M. Y.,
and Goldberg, A. L.
(1996)
Experientia Suppl. (Basel)
77,
57-78
15.
Lillie, S. H.,
and Pringle, J. R.
(1980)
J. Bacteriol.
143,
1384-1394
16.
Attfield, P. V.
(1987)
FEBS Lett.
225,
259-263
17.
Hottiger, T.,
Boller, T.,
and Wiemken, A.
(1987)
FEBS Lett.
220,
113-115
18.
Hottiger, T.,
De Virgilio, C.,
Hall, M. N.,
Boller, T.,
and Wiemken, A.
(1994)
Eur. J. Biochem.
219,
187-193
19.
Singer, M. A.,
and Lindquist, S.
(1998)
Trends Biotechnol.
16,
460-468
20.
Hengge-Aronis, R.,
Klein, W.,
Lange, R.,
Rimmele, M.,
and Boos, W.
(1991)
J. Bacteriol.
173,
7918-7924
21.
De Virgilio, C.,
Hottiger, T.,
Dominguez, J.,
Boller, T.,
and Wiemken, A.
(1994)
Eur. J. Biochem.
219,
179-186
22.
Lee, D. H.,
and Goldberg, A. L.
(1998)
Mol. Cell. Biol.
18,
30-38
23.
Singer, M. A.,
and Lindquist, S.
(1998)
Mol. Cell
1,
639-648
24.
Guo, N.,
Puhlev, I.,
Brown, D. R.,
Mansbridge, J.,
and Levine, F.
(2000)
Nat. Biotechnol.
18,
168-171
25.
Eroglu, A.,
Russo, M. J.,
Bieganski, R.,
Fowler, A.,
Cheley, S.,
Bayley, H.,
and Toner, M.
(2000)
Nat. Biotechnol.
18,
163-167
26.
Wieser, R.,
Adam, G.,
Wagner, A.,
Schuller, C.,
Marchler, G.,
Ruis, H.,
Krawiec, Z.,
and Bilinski, T.
(1991)
J. Biol. Chem.
266,
12406-12411
27.
Collinson, L. P.,
and Dawes, I. W.
(1992)
J. Gen. Microbiol.
138,
329-335
28.
Davies, J. M.,
Lowry, C. V.,
and Davies, K. J.
(1995)
Arch. Biochem. Biophys.
317,
1-6
29.
Wiese, A. G.,
Pacifici, R. E.,
and Davies, K. J.
(1995)
Arch. Biochem. Biophys.
318,
231-240
30.
Costa, V.,
Reis, E.,
Quintanilha, A.,
and Moradas-Ferreira, P.
(1993)
Arch. Biochem. Biophys.
300,
608-614
31.
Lee, J.,
Dawes, I. W.,
and Roe, J. H.
(1995)
Microbiology
141,
3127-3132
32.
Godon, C.,
Lagniel, G.,
Lee, J.,
Buhler, J. M.,
Kieffer, S.,
Perrot, M.,
Boucherie, H.,
Toledano, M. B.,
and Labarre, J.
(1998)
J. Biol. Chem.
273,
22480-22489
33.
Takahashi, R.,
and Goto, S.
(1990)
Arch. Biochem. Biophys.
277,
228-233
34.
Li, S.,
Patapoff, T. W.,
Nguyen, T. H.,
and Borchardt, R. T.
(1996)
J. Pharm. Sci.
85,
868-672
35.
Bell, W.,
Sun, W.,
Hohmann, S.,
Wera, S.,
Reinders, A.,
De Virgilio, C.,
Wiemken, A.,
and Thevelein, J. M.
(1998)
J. Biol. Chem.
273,
33311-33319
36.
Kienle, I.,
Burgert, M.,
and Holzer, H.
(1993)
Yeast
9,
607-611
37.
Cavicchioli, R.,
and Watson, K.
(1986)
FEBS Lett.
207,
149-152
38.
Bell, W.,
Klaassen, P.,
Ohnacker, M.,
Boller, T.,
Herweijer, M.,
Schoppink, P.,
Van der Zee, P.,
and Wiemken, A.
(1992)
Eur. J. Biochem.
209,
951-959
39.
De Virgilio, C.,
Burckert, N.,
Bell, W.,
Jeno, P.,
Boller, T.,
and Wiemken, A.
(1993)
Eur. J. Biochem.
212,
315-323
40.
Hazell, B. W.,
Nevalainen, H.,
and Attfield, P. V.
(1995)
FEBS Lett.
377,
457-460
41.
Sanchez, Y.,
Taulien, J.,
Borkovich, K. A.,
and Lindquist, S.
(1992)
EMBO J.
11,
2357-2364
42.
Tauber, A. I.,
and Babior, B. M.
(1977)
J. Clin. Invest.
60,
374-379
43.
Litchfield, W. J.,
and Wells, W. W.
(1978)
Arch. Biochem. Biophys.
188,
26-30
44.
Levine, R. L.,
Williams, J. A.,
Stadtman, E. R.,
and Shacter, E.
(1994)
Methods Enzymol.
233,
346-357
45.
Tamarit, J.,
Cabiscol, E.,
and Ros, J.
(1998)
J. Biol. Chem.
273,
3027-3032
46.
Cabiscol, E.,
Piulats, E.,
Echave, P.,
Herrero, E.,
and Ros, J.
(2000)
J. Biol. Chem.
275,
27393-27398
47.
Sitte, N.,
Merker, K.,
and Grune, T.
(1998)
FEBS Lett.
440,
399-402
48.
Elliott, B.,
Haltiwanger, R. S.,
and Futcher, B.
(1996)
Genetics
144,
923-933
49.
Gounalaki, N.,
and Thireos, G.
(1994)
EMBO J.
13,
4036-4041
50.
Chatuverdi, V.,
Wong, B.,
and Newman, S. L.
(1996)
J. Immunol.
156,
3836-3840
51.
Jennings, D. B.,
Ehrenshaft, M.,
Pharr, D. M.,
and Williamson, W. J.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
15129-15133W. J.
52.
Rego, A. C.,
Duarte, E. P.,
and Oliveira, C. R.
(1996)
Free Radic. Biol. Med.
20,
175-187
53.
Mizisin, A. P.,
Li, L.,
Perello, M.,
Freshwater, J. D.,
Kalichman, M. W.,
Roux, L.,
and Calcutt, N. A.
(1996)
Am. J. Physiol.
270,
90-97
54.
Mizisin, A. P.,
Li, L.,
and Calcutt, N. A.
(1997)
Neurosci. Lett.
229,
53-56
55.
Davies, K. J.,
and Goldberg, A. L.
(1987)
J. Biol. Chem.
262,
8227-8234
56.
Pacifici, R. E.,
Kono, Y.,
and Davies, K. J.
(1993)
J. Biol. Chem.
268,
15405-15411
57.
Giulivi, C.,
Pacifici, R. E.,
and Davies, K. J.
(1994)
Arch. Biochem. Biophys.
311,
329-341
58.
Grune, T.,
Reinheckel, T.,
and Davies, K. J.
(1997)
FASEB J.
11,
526-534
59.
Beers, R. F.,
and Sizer, I. W.
(1952)
J. Biol. Chem.
195,
133-140
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