(Received for publication, May 3, 1995; and in revised form, November 2, 1995)
From the
The 90-kDa stress protein, HSP90, is a major cytosolic protein
ubiquitously distributed in all species. Using two substrate proteins,
dihydrofolate reductase (DHFR) and firefly luciferase, we demonstrate
here that HSP90 newly acquires a chaperone activity when incubated at
temperatures higher than 46 °C, which is coupled with
self-oligomerization of HSP90. While chemically denatured DHFR refolds
spontaneously upon dilution from denaturant, oligomerized HSP90 bound
DHFR during the process of refolding and prevented it from
renaturation. DHFR was released from the complex with HSP90 by
incubating with GroEL/ES complexes in an ATP-dependent manner and
refolded into the native form. -Casein inhibited the binding of
DHFR to HSP90 and also chased DHFR from the complex with HSP90. These
results suggest that HSP90 binds substrates to maintain them in a
folding-competent structure. Furthermore, we found that HSP90 prevents
luciferase from irreversible thermal denaturation and enables it to
refold when postincubated with reticulocyte lysates. This heat-induced
chaperone activity of HSP90 associated with its oligomerization may
have a pivotal role in protection of cells from thermal damages.
Stress response and acquired stress tolerance are commonly seen in all organisms from bacteria to higher vertebrates and are considered to be essential defense mechanisms of the cell against various environmental stresses(1) . When cultured mammalian cells are exposed to 45 °C or higher temperatures, they are killed rapidly. However, if they are preheated at nonlethally high temperatures, they can survive the subsequent exposure to 45 °C(2, 3) . These results were interpreted as indications that the acquisition of thermotolerance is attributed to the accumulation of stress proteins. This has been supported by the fact that overexpression of stress proteins such as HSP90(3) , HSP70(4) , and HSP27 (5, 6, 7, 8) rendered cells stress-tolerant. In the case of budding yeast, HSP104 plays a crucial role in stress tolerance, and mutations in this gene drastically reduced survival at extreme temperatures(9) .
It is now accepted that stress proteins prevent catastrophic protein aggregation induced by heat and other stresses and also assist refolding of damaged proteins during recovery from stress(10, 11, 12, 13) . For instance, Escherichia coli molecular chaperones, DnaK/DnaJ/GrpE(14, 15, 16) , GroEL/ES(16, 17) , and ClpA(18) , mitochondrial HSP60(19) , and HSP90 (20) protect substrate proteins from thermally induced aggregation. Furthermore it was revealed that HSP104 does not prevent protein aggregation but rather disaggregates and reactivates stress-damaged proteins(21, 22) . DnaK/DnaJ/GrpE and GroEL/ES are also proposed to have reactivation activities(14, 16) .
HSP90 interacts with a specialized class of proteins such as steroid hormone receptors, protein kinases, and cytoskeletal proteins, and regulates their functions(11, 23) . Since the mode of action of HSP90 has not been fully investigated, its function as a molecular chaperone remains elusive. It has been shown recently, however, that HSP90 functions as a molecular chaperone to prevent protein aggregation (24, 25) and assist refolding of denatured proteins(26) . The endoplasmic reticulum homologue of HSP90, Grp94, was shown to mediate folding and assembly of immunoglobulin chains(27) .
We report here that, distinct from these chaperone activities expressed under normal conditions, HSP90 acquires another chaperone activity, which is latent under normal conditions but is induced by heating, to bind substrates and prevent their irreversible aggregation.
Figure 5:
Effects of -casein or R-CMLA on
inhibition of DHFR refolding by HSP90 preheated with
-peptide. A,
-casein or R-CMLA at the indicated concentrations was
mixed with buffer solutions with or without HSP90 preheated with
-peptide and incubated at 25 °C for 2 min. After adding
denatured DHFR, the activity of DHFR was measured. In each pair of
experiments with and without HSP90 at the same concentrations of
-casein or R-CMLA, inhibition of DHFR refolding is expressed as
percent of the activity obtained without HSP90. The activity of DHFR in
the absence of HSP90 was not altered by addition of
-casein or
R-CMLA (data not shown). B, denatured DHFR was diluted into
buffer solutions with or without HSP90 preheated with
-peptide and
incubated at 25 °C for 2 min. After adding
-casein or R-CMLA
at the indicated concentrations, DHFR activity was measured. Inhibition
of DHFR refolding is expressed as described
above.
Figure 2:
Binding of S-labeled DHFR to
oligomerized HSP90. GdmCl-denatured
S-labeled DHFR was
diluted into buffer solutions containing HSP90 at varying
concentrations (lanes 1 and 5, 1 µM; lanes 2 and 6, 2 µM; lanes 3, 4, 7, and 8, 4 µM) and heated
at 49 °C (lanes 1-3 and 5-7) or left
on ice (lanes 4 and 8) for 2 min. The mixtures were
resolved by native PAGE, and the gel was stained with Coomassie
Brilliant Blue (lanes 1-4), followed by autoradiography (lanes 5-8). Dimeric (2-mer) to octameric (8-mer) forms of HSP90 are indicated on the
left.
In
order to test this hypothesis, we have first analyzed the interaction
between HSP90 and denatured DHFR. It is well known that GdmCl-denatured
DHFR spontaneously refolds upon dilution from
denaturant(34, 35) . Recovery of the activity was
significantly reduced when denatured DHFR was diluted into buffer
solutions containing HSP90 at temperatures higher than 46 °C (Fig. 1A, lanes 4 and 6). When HSP90
was omitted, DHFR activity was fully recovered even when incubated at
49 °C (Fig. 1A, lanes 1, 3, and 5). In addition, bovine serum albumin did not affect DHFR
refolding at 49 °C (Fig. 1A, lane 7). The
inhibitory effect of HSP90 on the renaturation of DHFR observed at 49
°C was concentration-dependent, while no inhibition was observed at
25 °C (Fig. 1B). These results suggest that
unfolded DHFR could be bound to HSP90, which is probably converted to
oligomeric forms, thereby leading to prevention of DHFR refolding. To
examine this possibility, DHFR was labeled with
[S]methionine, and its binding to HSP90 was
analyzed by native PAGE using GmdCl-denatured
S-labeled
DHFR. As demonstrated before(33) , incubation of HSP90 at 49
°C induced self-oligomerization (Fig. 2, lanes
1-3). The proportion of oligomers was evidently dependent
upon the concentration of HSP90. We found that
S-labeled
DHFR was bound to oligomerized HSP90 but not to HSP90 dimers (Fig. 2, lanes 5-7). It is detectable for the
bands of HSP90 tetramers (4-mer in Fig. 2) that the radioactive
bands slightly more slowly migrated compared with the Coomassie
Brilliant Blue-stained bands, which resulted from DHFR binding to HSP90
tetramers (Fig. 2, compare lanes 1-3 with lanes 5-7, respectively). When the mixtures were kept on
ice, neither HSP90 oligomerization nor DHFR binding to HSP90 occurred (Fig. 2, lanes 4 and 8). Once HSP90
oligomerized by heating, the oligomeric forms stably remained even
after the temperature was lowered. ATP did not affect the
oligomerization of HSP90 (data not shown). It should be mentioned that
HSP90, which had been incubated at 49 °C for 2 min and then cooled
to 25 °C, was still oligomeric but unable to prevent DHFR refolding
(data not shown; cf. Fig. 4).
Figure 1: Heat-induced activity of HSP90 to prevent DHFR refolding. A, GdmCl-denatured DHFR (22 nM) was diluted into buffer solutions in the absence (lanes 1, 3, and 5) or presence (lanes 2, 4, and 6) of 2.2 µM HSP90 and incubated at 43, 46, and 49 °C for 2 min followed by measurement of DHFR activity that is expressed as percent of the recovered activity when incubated at 25 °C for 2 min without HSP90. GdmCl-denatured DHFR was diluted into a buffer solution containing 2 µM bovine serum albumin and incubated at 49 °C for 2 min (lane 7). B, GdmCl-denatured DHFR (22 nM) was diluted into buffer solutions containing HSP90 at indicated concentrations and incubated at 25 or 49 °C for 2 min, followed by measurement of DHFR activity. The activity of DHFR spontaneously refolded at 25 °C for 2 min was set as 100%.
Figure 4:
Prevention of DHFR refolding by HSP90
preheated with -peptide. GdmCl-denatured DHFR was diluted at 25
°C into buffer solutions containing the indicated concentrations of
HSP90, which had been preheated at 43 °C for 40 min in the presence
or absence of
-peptide and then cooled to 25 °C, after which
DHFR activity was measured. The activity of DHFR refolded in the
absence of HSP90 was set as 100%.
Figure 3: Refolding of DHFR complexed with HSP90 by GroEL/ES. GdmCl-denatured DHFR (22 nM) was diluted into buffer solutions without (lane 1) or with (lanes 2-4) HSP90 (2.2 µM) and incubated at 49 °C for 2 min, followed by incubation at 25 °C for 2 min in the presence or absence of 1 µM GroEL/ES complexes. After addition of 1 mM ATP when indicated, DHFR activity was measured. The activity obtained for lane 1 is set as 100%.
We have previously shown that the synthetic 21-residues
peptide corresponding to the calmodulin-binding domain of mouse
HSP90,
-peptide, is able to bind to HSP90 itself and to
stimulate the self-oligomerization(33) . When GdmCl-denatured
DHFR was diluted at 25 °C into buffer solutions containing HSP90
that had been preincubated at 43 °C for 40 min in the presence of
-peptide and cooled to 25 °C, DHFR refolding was significantly
prevented (Fig. 4). Furthermore the inhibitory activity of HSP90
preheated with
-peptide lasted at least for 2 h even after cooled
to 25 °C (data not shown), indicating that HSP90 may be fixed in a
binding-competent state for substrates by preheating with
-peptide
(see ``Discussion''). HSP90 (Fig. 4) or
-peptide
(data not shown) preheated alone did not exhibit such an inhibitory
activity on DHFR refolding.
When -casein, which has properties
of partially denatured proteins in its native state (35, 36) and is bound to
GroEL(37, 38) , was added to the solutions of HSP90
preheated with
-peptide before mixing with denatured DHFR, the
inhibitory effect of HSP90 was remarkably reduced in a manner dependent
on concentrations of
-casein (Fig. 5A). This
suggests that the binding of
-casein to HSP90 competitively
prevented access of DHFR to HSP90. Moreover, postaddition of
-casein to the mixtures containing complexes of DHFR and HSP90
caused significant reduction of the inhibitory effect of HSP90, which
is due to release and renaturation of DHFR (Fig. 5B).
Thus
-casein, which does not have a chaperone activity on its own,
seems to release DHFR bound to HSP90, probably by replacing DHFR on the
HSP90 molecule because it has affinity to HSP90 as shown in Fig. 5A. In contrast to
-casein, R-CMLA, which
exists in an extended form in aqueous solutions without
detergent(38, 39, 40) , did not affect the
inhibition of DHFR refolding by HSP90, irrespective of whether its
addition was before or after addition of denatured DHFR (Fig. 5, A and B, lanes 6). Therefore HSP90 seems to
recognize a folding intermediate structure that is represented by
-casein, and DHFR bound to HSP90 is probably in such a structure.
Luciferase was completely inactivated upon incubation for 5 min at temperatures higher than 45 °C (Fig. 6, lanes 1 and 2). Incubation with reticulocyte lysate after thermal inactivation could not reactivate thermally damaged luciferase (Fig. 6, lanes 3 and 4). When HSP90 was present during thermal inactivation, luciferase could not be protected from denaturation (Fig. 6, lanes 5 and 6), but reactivation of luciferase was induced by postincubation with reticulocyte lysates after thermal inactivation (Fig. 6, lanes 7 and 8). Thus it is likely that HSP90 prevented irreversible denaturation of luciferase and maintained it in a folding-competent state during thermal inactivation, from which luciferase could be reactivated by the unidentified component(s) in reticulocyte lysates. Interestingly, the activity of luciferase was reproducibly higher at 50 than at 45 °C (Fig. 6, lanes 7 and 8). This may be explained by the following possibility that at higher temperatures, HSP90 is more rapidly converted to a form that is able to capture denatured proteins, thereby leading to more effective reactivation of luciferase.
Figure 6: Protection of luciferase from irreversible denaturation by HSP90. Luciferase (250 nM) was incubated at 45 or 50 °C for 5 min in buffer solutions with (+) or without(-) HSP90 (20 µM). After cooled to 25 °C, aliquots were incubated at 25 °C for 1 h with (+) or without(-) reticulocyte lysates and then luciferase activity was determined. The activity before thermal denaturation is set as 100%.
In this study, we demonstrated using two substrates,
chemically denatured DHFR and thermally denatured luciferase, that
HSP90 newly acquires a chaperone activity upon incubation at high
temperatures (Fig. 1A and Fig. 6). This
heat-induced chaperone activity is not detectable before heating at all (Fig. 1B) and is coupled with self-oligomerization (Fig. 2). DHFR bound to oligomerized HSP90 was released and
refolded by GroEL/ES in the presence of ATP (Fig. 3).
Furthermore -casein chased bound DHFR from the HSP90 molecule to
cause its spontaneous refolding (Fig. 5B). On the other
hand, the HSP90 oligomers were not dissociated in either case. (
)
-Casein, which possesses a folding intermediate-like
structure in its native state(35, 36) , inhibited the
binding of DHFR to oligomerized HSP90 (Fig. 5A),
probably because HSP90 recognized a structure of folding intermediates
and bound
-casein. Thus these results exclude the trivial
possibility that DHFR binding to oligomerized HSP90 was a consequence
of co-aggregation of the two proteins and indicate that oligomerized
HSP90 keeps DHFR in a folding-competent state.
HSP90 rapidly lost
the newly acquired chaperone activity to bind denatured DHFR while
HSP90 oligomers stably remained when it was cooled to 25 °C after
heating. Thus it should be emphasized that oligomerized
HSP90 per se is not responsible for the novel chaperone
activity but that conformational changes of HSP90 leading to its
oligomerization are required for induction of the chaperone activity.
Both self-oligomerization and the novel chaperone activity are
suggested to be attributable to the same conformational changes induced
on HSP90 by heat. In fact, the conformation of HSP90 alters upon
heating with an increase of
hydrophobicity(44, 45, 46) . Although HSP70
is also reported to undergo oligomerization at high
temperatures(39) , it is unclear whether the chaperone activity
of HSP70 is facilitated. Analysis by native PAGE revealed that DHFR is
bound exclusively to oligomerized HSP90 larger than tetramers but not
to dimeric forms (Fig. 2). It would be possible that these
dimers might not efficiently undergo conformational changes. Therefore
they remained dimers and could not bind denatured DHFR.
Although the
activated state of HSP90 is labile unless the temperature is left high,
when HSP90 is incubated in the presence of -peptide, which
corresponds to the calmodulin-binding domain of mouse HSP90
(33) , the novel chaperone activity of HSP90 to capture
denatured DHFR is prolonged at least for 2 h even after cooled to 25
°C.
Furthermore, this chaperone activity was induced at
43 °C (Fig. 4). As discussed previously, HSP90 probably has
an intrinsic site interacting with its own calmodulin-binding domain
and therefore
-peptide can bind to HSP90(33) . In
addition, this peptide enhances self-oligomerization of
HSP90(33) . Taken together, the hypothetical intramolecular
interaction between the calmodulin-binding domain and its counterpart
may be involved in a heat-induced process of HSP90 oligomerization.
Dissociation of this interaction is likely to be caused by heating,
thereby leading to conformational changes of HSP90 responsible for
self-oligomerization. If it is the case, it is conceivable that
-peptide can stimulate HSP90 oligomerization and stabilize
conformational changes of HSP90, which are labile otherwise, even after
cooling. Therefore HSP90 preheated with
-peptide can long retain
the activity to bind denatured DHFR.
When cells are exposed to high temperatures, a significant portion of proteins is denatured and tends to form aggregates, which are extremely harmful to cells. Following are two ways to protect cells from such a catastrophe: one is prevention of off-pathway reactions(14, 15, 16, 17, 18, 19, 20) , and the other is solubilization and reactivation of aggregated proteins (14, 16, 21, 22) . The former seems primarily important in protection of cells, since no matter how effective the latter system is, it cannot work if cells do not survive stress. Assuming that the duration of heat shock is prolonged to exhaust all the preexisting chaperones, more chaperones are necessary. To meet this demand, cells are equipped with a specific system of inducing the synthesis of chaperones, which were therefore called heat-shock (or stress) proteins at first(1, 47) . Furthermore, we propose here another system of inducing a novel chaperone activity without increase in the amount of chaperones. As seen above, HSP90 acquires the chaperone activity to bind partially unfolded proteins, which is latent under normal conditions but appears upon incubation at elevated temperatures. Although HSP90 can bind and protect proteins from irreversible aggregation, it cannot release and reactivate the bound proteins without assistance of other chaperones. Since what is required in an emergency is such an action, the heat-induced activity of HSP90 appears quite important for a defense system of the cell. E. coli chaperone ClpA was reported to have a similar activity toward thermally denatured luciferase in which DnaK/DnaJ/GrpE can reactivate luciferase bound to ClpA(18) . Although we have found in this study that reticulocyte lysates can reactivate luciferase captured by HSP90, the factor(s) in lysates has not been identified yet. Since GroEL/ES complexes can release DHFR bound to HSP90 as shown above, the functionally equivalent eukaryotic chaperonin TRiC (11, 13) might be the most likely candidate for the factor, which remains to be tested.