(Received for publication, November 3, 1994; and in revised form, December 5, 1994)
From the
The heat-induced expression of heat shock proteins, called the cellular stress response, is mediated by heat shock transcription factor 1 (HSF1). HSF1 exists in unstressed cells in an inactive form, which is converted to the DNA binding form upon exposure of cells to elevated temperature. We have developed a protocol for isolation of the non-DNA binding form of recombinant mouse HSF1, involving expression and affinity purification of HSF1 as a fusion with the glutathione S-transferase protein in Escherichia coli, followed by specific protease cleavage to release pure HSF1 protein. We report here that the purified inactive HSF1 can be converted to the DNA binding form by heat treatment in vitro. Chemical cross-linking analysis demonstrates that this conversion is accompanied by oligomerization of HSF1 from a monomeric to a trimeric native structure, similar to that observed for HSF1 in heat-shocked cells. These results indicate that elements residing in the HSF1 polypeptide are sufficient both for maintenance of this factor in the non-DNA binding form and for its heat-induced conversion to the DNA binding form and support a role for HSF1 as the ``molecular thermostat'' in eukaryotic cells, which senses adverse environmental conditions and activates the cellular stress response.
Cells exposed to elevated temperature and other environmental
stress conditions respond by rapidly inducing the expression of heat
shock proteins (hsps), ()which function to protect cells
from the harmful effects of stress conditions on cellular proteins
(reviewed in (1, 2, 3) ). The induction of
the cellular stress response is mediated by heat shock transcription
factor 1 (HSF1), which exists in unstressed cells in a monomeric
non-DNA binding form and is converted to the trimeric DNA binding form
following exposure of cells to elevated temperature and other stress
conditions (4, 5, 6) (reviewed in (7, 8, 9, 10) ).
One important
question regarding the regulation of the cellular stress response is
the mechanism by which HSF1 is maintained in the non-DNA binding form
in unstressed cells. A C-terminal leucine zipper motif in human HSF1
has been shown to be important for maintaining this factor in the
non-DNA binding form, apparently via its interactions with the
N-terminal leucine zipper oligomerization domain, thus inhibiting the
trimerization of HSF1, which is a prerequisite for DNA binding activity (11, 12) . These results suggest that the mechanism
that controls HSF1 DNA binding is inherent in the structure of the HSF1
polypeptide. However, a number of observations have suggested that
other molecules may be involved in regulating HSF1 DNA binding
activity. Overexpression of HSF1 in mouse NIH 3T3 cells results in
constitutive trimerization and DNA binding activity of this factor,
supporting a role for a titratable negative regulator in controlling
HSF1 activity(4) . Several studies have suggested that hsp70
may be involved in regulating HSF1 DNA binding activity, providing
support for the existence of a negative feedback loop for the
regulation of the cellular stress response (13, 14, 15, 16, 17, 18) .
Evidence exists for a similar mechanism for control of bacterial heat
shock protein expression, involving interaction of dnaK, dnaJ, and grpE
with the bacterial heat shock transcription factor
-32(19, 20, 21, 22) . However,
the results of a recent study, while supporting a role for hsp70 in
deactivation of HSF trimers during attenuation of the cellular stress
response, failed to observe any effect of hsp70 on either the
temperature set point or magnitude of HSF activation(23) .
A second fundamental question is the identity of the ``cellular thermometer'' responsible for sensing elevated temperature and the nature of the signal transduction mechanism that converts this sensing event into activation of HSF1 DNA binding (reviewed in Refs. 14 and 15). Although a number of hypotheses have been put forward concerning this mechanism, the central question of whether HSF1 can directly sense elevated temperature or whether another molecule serves as the sensor and then relays the signal to HSF1 has not been answered.
In order to directly address these questions, we have devised a protocol for purification of the non-DNA binding form of recombinant mouse HSF1 following expression in Escherichia coli. We have shown that this purified HSF1 can be converted to the trimeric, DNA binding form by heat treatment in vitro. These results indicate that other proteins are not required for maintenance of HSF1 in the inactive form or for its conversion to the active DNA binding form in response to heat treatment. These results implicate HSF1 as the ``molecular thermostat'' of the cellular stress response, capable of sensing and responding to elevations in cellular temperature by oligomerization to the trimeric DNA binding form.
For purification of the GST-HSF1 fusion
protein, pellets were removed from -80 °C storage and
resuspended in 500 µl of ice-cold Buffer D (20 mM HEPES
(pH 7.9), 10% glycerol, 100 mM KCl, 0.2 mM EDTA). All
microcentrifuge tubes used in the purification protocol were silanized
by placing them under vacuum with a vial of dimethyldichlorosilane in
chloroform for 12 h. Resuspended cells were lysed by 2 5-s
pulses of sonication at 25% full energy (55 milliwatts) with 30 s of
chilling between pulses in an ice/ethanol bath using a model W220
ultrasonic cell disruptor from Heat Systems (Farmingdale, NY). After
pelleting insoluble material by centrifugation at 16,000
g for 3 min at 4 °C, a 200-µl aliquot of the extract was
diluted with 400 µl of PBS containing 0.2 M NaCl (PBS/0.2 M) and incubated with 200 µl of a 50% slurry of
glutathione-agarose beads (Sulfo-linked, Sigma) at 4 °C for 1 h
with constant inversion mixing. The glutathione-agarose beads were then
washed 3 times with 1 ml of ice-cold PBS/0.2 M and finally
resuspended in 100 µl of PBS/0.2 M supplemented with
CaCl
to a concentration of 5 mM. Thrombin (human,
Sigma) was then added to a concentration of 7.5 ng/µl (0.02
units/µl), and cleavage was carried out at 20 °C for 1 h with
constant inversion mixing. Cleavage of the fusion protein yields an
HSF1 polypeptide with the N-terminal sequence, N-Gly-Ser-Pro-Met-,
where Met is the beginning of the predicted natural HSF1
sequence(25) . Cleaved HSF1 polypeptide was separated by
centrifugation and used for the experiments described below.
We identified several parameters that are important for obtaining purified HSF1 in the non-DNA binding form. The first parameter is the growth temperature of the E. coli in which the GST-HSF1 fusion protein is expressed. From cells grown in an incubator with a chamber temperature of 33 °C, we were consistently able to obtain purified HSF1 in the non-DNA binding form, which could be activated to the DNA binding form by heat treatment in vitro. Growth at lower temperatures such as 30 °C gave similar results as for 33 °C, although lower levels of heat-inducible DNA binding activity were observed, perhaps due to lower amounts of fusion protein expression in these cells. Higher growth temperatures, such as 37 °C, typically yielded purified HSF1 that had significant constitutive DNA binding activity. Another important parameter is the method used to lyse the bacteria. The sonication conditions we employ released the highest levels of GST-HSF1 from the bacteria without causing the appearance of constitutive DNA binding activity. Conditions more rigorous than these, employing either higher energy bursts of sonication or bursts of longer duration, yielded significantly higher levels of constitutive DNA binding activity.
As discussed above, two important questions in the regulation of the cellular stress response are the mechanisms involved in the control and activation of HSF1 DNA binding in unstressed and heat-treated cells, respectively. In order to provide a system with which to directly examine these mechanisms, we set out to devise a protocol for purification of the non-DNA binding form of HSF1. We chose to express HSF1 as a fusion protein with GST in E. coli because this expression system yields high levels of protein in a readily purifiable form(24) . The GST-HSF1 fusion protein was expressed in E. coli grown at 33 °C, purified by affinity chromatography on glutathione-agarose, and then cleaved with thrombin while still bound to the glutathione-agarose, in order to release pure HSF1 protein while leaving the 26-kDa GST sequence on the affinity resin. Fig. 1shows that the purified HSF1 migrates on an SDS-PAGE gel as a single band of approximately 67 kDa.
Figure 1: Purification of HSF1 overexpressed in E. coli. HSF1 was expressed in E. coli as a GST-fusion protein (24) and purified as described under ``Experimental Procedures.'' The lane marked C was loaded with crude extract of bacteria expressing the GST-HSF1 fusion protein (position indicated by a opentriangle). The lane marked P was loaded with purified HSF1 protein (indicated by a closedtriangle). The sizes of molecular mass standards (in kDa) loaded in the lane marked M are indicated on the leftside of the panel.
We first wanted to determine whether the purified HSF1 existed in the non-DNA binding form, and if so, whether it could be converted to the DNA binding form by heat treatment in vitro, as has been shown for HSF1 in crude cytosolic extracts of non-heat-shocked mouse and human cells(26, 27) . Therefore, purified HSF1 protein was incubated at various temperatures for 30 min and then subjected to gel mobility shift assay. Because we could not predict which temperatures would induce the DNA binding activity of the purified HSF1, the initial analysis examined temperatures over a broad range. Fig. 2A shows the results obtained for HSF1 purified from bacteria grown at 30 °C. Little DNA binding activity was observed following incubation of purified HSF1 at 22 or 27 °C, but high levels of binding activity were induced by treatment at 32 °C, followed by diminishing levels in extracts incubated at 37 and 42 °C. Identical results were obtained using HSF1 protein purified from E. coli grown at a chamber temperature of 33 °C (Fig. 2B). Since we consistently obtained higher yields of purified protein from cells grown at 33 °C than cells grown at 30 °C, subsequent experiments were performed using HSF1 purified from bacteria grown at the higher temperature. To precisely determine the threshold temperature for activation of DNA binding, purified HSF1 (from the same cells used for the experiment shown in Fig. 2B) was incubated at temperatures between 30 and 35 °C, using 1 °C increments, and then subjected to gel shift analysis. Fig. 3shows that purified HSF1 exhibits a relatively sharp temperature profile of DNA binding activation, with an apparent threshold temperature of 32 °C, similar levels of DNA binding activity at 33 °C, and diminishing levels at 34 °C.
Figure 2:
Activation of purified HSF1 DNA binding by
heat treatment in vitro. Aliquots of recombinant HSF1 (20
ng/lane) purified from bacteria grown at 30 °C (panelA) or 33 °C (panelB) were
incubated at the indicated temperatures for 30 min and then subjected
to gel mobility shift analysis as described previously (4) using a P-end-labeled HSE-containing
oligonucleotide probe. The closedtriangles indicate
HSF1 DNA binding activity, and F indicates the position of
free HSE probe.
Figure 3: Threshold temperature for induction of purified HSF1 DNA binding activity. The experiment was performed as described in the legend to Fig. 2except that aliquots of purified HSF1 were incubated at temperatures varying by 1 °C increments between 30 and 35 °C. The closedtriangle indicates HSF1 DNA binding activity.
Activation of HSF1 DNA binding in heat-treated eukaryotic cells is accompanied by oligomerization of this factor from a monomeric form to a trimeric form(4, 5, 6, 28) . Therefore, to determine if purified HSF1 undergoes a similar change in oligomeric state during activation of DNA binding in vitro, purified protein was incubated at 22 or 32 °C for 30 min and then subjected to chemical cross-linking analysis using EGS. The results, shown in Fig. 4, demonstrate that no cross-linking products are observed in samples of purified HSF1 incubated at 22 °C. Identical results were obtained for HSF1 in extracts of unstressed mouse NIH 3T3 cells(4) , suggesting that the non-DNA binding form of purified recombinant HSF1, like its natural counterpart in mouse cells, is comprised primarily of monomeric HSF1. However, cross-linking of purified HSF1 incubated at 32 °C yields products whose sizes are consistent with dimeric (0.1 mM EGS) and trimeric (0.5 mM EGS) HSF1 complexes. These results are identical to those obtained for HSF1 in extracts of heat-shocked mouse NIH 3T3 cells(4) , indicating that activation of DNA binding of purified HSF1 is also accompanied by oligomerization to a trimeric native structure.
Figure 4: Heat-induced change in native size of purified HSF1. For analysis of heat-induced changes in the native size of purified recombinant HSF1, inactive (22 °C, 30 min) and activated (32 °C, 30 min) purified HSF1 (20 ng/reaction) was subjected to chemical cross-linking using either no EGS or 0.1 or 0.5 mM EGS at 22 °C for 30 min. HSF1 cross-linking products were separated by SDS-PAGE gel electrophoresis and detected by Western blot analysis using HSF1 polyclonal antibodies(4) . The sizes of molecular mass standards (in kDa) are indicated on leftside of the panel.
The results presented above suggest that elements residing in the primary amino acid sequence of the HSF1 polypeptide are sufficient both for maintenance of HSF1 in the monomeric non-DNA binding form and for activation of this factor to the trimeric DNA binding form in response to heat treatment. These data support a role for HSF1 as the ``molecular thermostat'' of the cellular stress response, a protein capable of sensing and responding to elevation of cellular temperature by undergoing oligomerization to the trimeric DNA binding form. Our findings are consistent with a previously postulated mechanism for HSF activation involving stress-induced disruption of an interaction between a C-terminally located sequence and the N-terminal oligomerization domain, thus allowing formation of the trimeric DNA binding form(11, 12) .
These results do not rule out the potential role of other proteins, most notably hsps, in formation of the inactive HSF1 state, in modulating the temperature set point, kinetics, or magnitude of activation of HSF1 DNA binding in eukaryotic cells, or in converting active trimeric HSF1 back to the inactive monomeric form during attenuation of the cellular stress response(16, 17, 18, 23) . In fact, the observed threshold temperature of 32 °C for activation of purified HSF1 DNA binding indicates that other factors must be involved in modulating the temperature set point of HSF1 activation in eukaryotic cells, which typically occurs at temperatures above 41 °C. One simple explanation is that the protein constituency of eukaryotic cells creates a biochemical environment that stabilizes HSF1 conformation, so that higher temperatures are required to trigger the activation process. This possibility is consistent with results demonstrating that HSF activation temperature is modulated by the biochemical environment of the cell in which it is expressed(29) .
Although our results are consistent with a mechanism in which heat directly acts on the HSF1 protein to activate its DNA binding activity, they are not inconsistent with other potential mechanisms for HSF1 activation in eukaryotic cells. For example, there is substantial evidence supporting the hypothesis that stress-induced denaturation of proteins, and not heat perse, is the signal that triggers HSF1 activation(30, 31, 32, 33) . How could protein denaturation cause HSF1 activation? One possibility is that protein hydrophobic domains exposed during heat-induced protein denaturation directly act on HSF1 to convert it to the DNA binding form. Since leucine zipper interactions such as those proposed to maintain HSF1 in the inactive monomeric form (11, 12) are driven by unfavorable interactions of these hydrophobic amino acid sequences with water molecules, the HSF1 zipper interaction could be disrupted by alterations in local water structure surrounding the HSF1 protein caused by reordering of water molecules around nearby exposed protein hydrophobic domains. Alternatively, the HSF1 zipper interaction could be disrupted by transient competitive hydrophobic interactions between the exposed protein hydrophobic domains and either of the two HSF1 leucine zipper sequences. These potential mechanisms are supported by previous results demonstrating that two substances known to strengthen protein hydrophobic interactions, deuterium oxide and glycerol, are able to inhibit induction of the cellular stress response(27, 34, 35, 36) .
Previous studies have shown that HSF1 expressed in E. coli exhibits constitutive trimerization and DNA binding activity(4, 37) . One possible explanation for this result is that the high concentration of recombinant protein being synthesized in these cells allows the oligomerization domains of nascent HSF1 polypeptides to interact before the entire HSF1 protein has time to fold into an inactive, non-DNA binding state. This possibility is consistent with results described above supporting the function of an interaction between a sequence near the C terminus of HSF1 and the N-terminal oligomerization domain in controlling the ability of this factor to trimerize (and, therefore, to bind DNA)(11, 12) . Because we expressed HSF1 in E. coli as a fusion with the GST polypeptide, it may be that the GST protein sequence inhibits premature trimerization by sterically hindering the interaction of nascently translated HSF1 oligomerization domains, thus allowing the entire polypeptide time to fold properly into the non-DNA binding form. Not surprisingly, we found that the growth temperature of the E. coli in which the GST-HSF1 fusion protein is expressed is also an important factor for obtaining HSF1 in the non-DNA binding form. Expression of the protein in cells grown at 33 °C yielded the best results, while growth at 37 °C consistently yielded HSF1 in the trimeric, DNA binding form. It is possible that the biochemical environment of E. coli is such that HSF1 expressed in cells grown at 33 °C, but not 37 °C, is able to fold properly into the non-DNA binding form.
The ability to activate purified HSF1 by heat treatment in vitro will provide a system with which to perform physical studies on the structural changes that occur in the HSF1 protein as it is converted from the inactive monomeric form to the trimeric, DNA binding form. These studies should yield interesting insights into the mechanism of HSF1 activation and further our understanding of the functional role of the HSF1 protein as both sensor and transcriptional effector in an important pathway of environmental signal transduction, the cellular stress response.