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Originally published In Press as doi:10.1074/jbc.M101487200 on April 11, 2001

J. Biol. Chem., Vol. 276, Issue 26, 24261-24267, June 29, 2001
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Trehalose Accumulation during Cellular Stress Protects Cells and Cellular Proteins from Damage by Oxygen Radicals*

Nadia BenaroudjDagger, Do Hee Lee§, and Alfred L. Goldberg

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (alpha -D-glucopyranosyl-1,1-alpha -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).

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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Saccharomyces cerevisiae Strains-- The ise1 strain JN284: MATalpha 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 tps1Delta ::TRP1 tps2Delta ::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.

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.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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).


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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.

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).


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  		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).

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.

                              
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Table I
Catalase activity and trehalose content in wild-type and trehalose mutant cells
W303-1a and tps1tps2 mutant cells were grown at 25 °C until mid-log phase and then transferred to 35 °C or maintained at 25 °C for 30 min. Cells were collected, and catalase activity and trehalose content were determined. Catalase activity was assayed spectrophotometrically by following the disappearance of H2O2 at 240 nm in 75 µg of yeast extract (59). The data for catalase activity are the means of three independent experiments. The data for trehalose content are the means of two independent experiments. WT, wild type.


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  		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.

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.


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  		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.

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.


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  		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."

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).


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  		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.

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.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (alpha -D-glucopyranosyl-1,2-beta -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.

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.

    ACKNOWLEDGEMENTS

We thank J. Thevelein and B. Futcher for providing the yeast strains.

    FOOTNOTES

* 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.

Dagger Supported by postdoctoral fellowships from Association pour la Recherche contre le Cancer and Human Frontier Science Program Organization.

§ 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

    ABBREVIATIONS

The abbreviations used are: ROS, reactive oxygen species; HSP, heat shock proteins; TPS1, trehalose-6-phosphate synthase; TPS2, trehalose-6-phosphate phosphatase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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