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J. Biol. Chem., Vol. 275, Issue 28, 21107-21113, July 14, 2000
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From the
Received for publication, February 16, 2000, and in revised form, April 27, 2000
Classic in vitro studies show that
the Hsp70 chaperone system from Escherichia coli
(DnaK-DnaJ-GrpE, the DnaK system) can bind to proteins, prevent
aggregation, and promote the correct refolding of chaperone-bound
polypeptides into native proteins. However, little is known about how
the DnaK system handles proteins that have already aggregated. In this
study, glucose-6-phosphate dehydrogenase was used as a model system to
generate stable populations of protein aggregates comprising controlled
ranges of particle sizes. The DnaK system recognized the
glucose-6-phosphate dehydrogenase aggregates as authentic substrates
and specifically solubilized and refolded the protein into a native
enzyme. The efficiency of disaggregation by the DnaK system was high
with small aggregates, but the efficiency decreased as the size of the
aggregates increased. High folding efficiency was restored by either
excess DnaK or substoichiometric amounts of the chaperone ClpB. We
suggest a mechanism whereby the DnaK system can readily solubilize
small aggregates and refold them into active proteins. With large
aggregates, however, the binding sites for the DnaK system had to be
dynamically exposed with excess DnaK or the catalytic action of ClpB
and ATP. Disaggregation by the DnaK machinery in the cell can
solubilize early aggregates that formed accidentally during
chaperone-assisted protein folding or that escaped the protection of
"holding" chaperones during stress.
A network of molecular chaperones in the cell controls the correct
folding of nascent and translocating polypeptides, the stability of
native proteins under stress, and the refolding of denatured proteins
following stress (for reviews see Refs. 1 and 2). Chaperones can
specifically recognize and interact with non-native proteins, mostly
through hydrophobic interactions with exposed hydrophobic surfaces.
Some chaperones such as HtpG, DnaK, DnaJ, GroEL, and IbpB from
Escherichia coli are also termed "holders" because they
can form a binary complex with unstable protein folding intermediates
and thus prevent aggregation (for a review see Ref. 3). However, under
physiological conditions chaperones such as GroEL in the presence of
GroES and ATP or DnaK in the presence of DnaJ, GrpE, and ATP can also
act as "folders," which interact in a more dynamic manner with
protein folding intermediates and thus maintain proteins within folding
pathways that lead to the native structure. Much remains to be
understood about the molecular mechanism through which different
chaperone systems assist protein folding. Cycles of binding and release
that are fueled by ATP and hydrolysis may be a common theme in the mode of action of chaperones that are as structurally and functionally different as DnaK, GroEL, and HtpG (2).
Problems arise during stress when unfolded proteins become highly
unstable and escape the protective action of the "holding" reservoir of the chaperone network. Soluble unfolded proteins do not
remain in solution waiting for assisted refolding by "folding" chaperones after stress. Rather, unfolded proteins tend to assume an
alternatively stable conformation in the form of insoluble aggregates
enriched with In vitro, most chaperone systems are inefficient in actively
dissolving protein aggregates. For the DnaK system, only one report
mentions that a molar excess of the DnaK system can efficiently reactivate previously heat-aggregated RNA polymerase (6, 7). Negligible
amounts of pre-aggregated enzymes such as luciferase are recovered in
the presence of a large (100-1000-fold) molar excess of the DnaK or
Hsp70 chaperone systems (8-11). In contrast, substoichiometric levels
of the DnaK system in the presence of the chaperone ClpB suffice to
efficiently solubilize and reactivate a wide array of previously
aggregated E. coli and model protein substrates (12, 13).
ClpB was shown to modify and precondition the nature of large turbid
aggregates toward subsequent solubilization and refolding by the DnaK
system (12). However, the molecular mechanism by which the DnaK system
alone mediates efficient solubilization of protein aggregates and the
spectrum of action of the DnaK system with regard to the nature and
size of the aggregated substrates remain unclear.
In this study, we address the mechanism by which the DnaK chaperone
system achieves solubilization and refolding of protein aggregates that
have different degrees of complexity. Using stable forms of aggregates
of glucose-6-phosphate dehydrogenase
(G6PDH)1 as model substrates,
we have demonstrated that the DnaK system alone can directly interact,
disaggregate, and reactivate stably aggregated protein particles. The
efficiency of disaggregation decreased as the size of the aggregate
increased, but efficient disaggregation was restored by a large molar
excess of DnaK or catalytic amounts of ClpB.
Proteins--
Protein purifications were performed according to
published procedures: DnaK, DnaJ, and GrpE (14), ClpB (15), GroEL and GroES (16), and IbpB (17). Rabbit muscle pyruvate kinase and lyophilized G6PDH from Leuconostoc mesenteroides were
purchased from Sigma. G6PDH was resuspended in 20% glycerol to a final
concentration of 300 µM and used as a stock solution.
Protein concentrations were determined using the Bio-Rad Bradford assay
with bovine serum albumin as a standard. Protein concentrations are
always expressed in protomers.
G6PDH Denaturation--
Various concentrations of native G6PDH
were denatured in three steps: (i) unfolding (5 min at 47 °C in 5 M urea, 20 mM dithiothreitol, and 8%
glycerol); (ii) dilution (50-fold dilution in folding buffer (50 mM triethanolamine, pH 7.5, 20 mM
MgAc2, 150 mM KCl, 10 mM dithiothreitol, and 3 mM phosphoenolpyruvate) at 47 °C);
(iii) stabilization (15 min at 47 °C). Large turbid G6PDH aggregates were formed by a 20 min incubation in folding buffer at 65 °C.
Size-exclusion Chromatography--
The oligomeric state and
apparent size of the various forms of denatured G6PDH were estimated by
size-exclusion chromatography in buffer (50 mM
triethanolamine, pH 7.5, 20 mM MgAc2 and 150 mM KCl), using a Superose 6HR10/30 gel filtration column
(Amersham Pharmacia Biotech) at a flow rate of 0.5 ml/min. The
absorbance was monitored at 280 nm. Apparent molecular weights were
estimated by gel filtration standards (Bio-Rad).
Spectroscopy--
Spectroscopic measurements were performed in a
Perkin-Elmer luminescence spectrometer LS50B. The various forms and
concentrations of aggregated G6PDH were incubated in folding buffer
with 50 µM 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic
acid (bis-ANS, Molecular Probes) for 5 min. bis-ANS fluorescence of
G6PDH was measured with excitation at 397 nm and emission at 496 nm.
Turbidity was measured in a four-sided quartz cuvette at an excitation
and emission wavelength of 550 nm.
Chaperone Activity Assay--
Assays were performed in folding
buffer in the presence of an ATP-regenerating system (3 mM
phosphoenolpyruvate and 20 µg/ml pyruvate kinase), which was active
for the concentration chaperones tested over at least 5 h at
30 °C. Unless otherwise specified, concentrations and relative
chaperone ratios in the DnaK system remained constant (3.5 µM DnaK, 0.7 µM DnaJ, 0.35 µM
GrpE). Refolding of G6PDH was initiated at 30 °C by the addition of
3 mM ATP and pyruvate kinase. Maximal rates of G6PDH
refolding were derived from the linear phase of the time curves of
recovered enzymatic activity (18). Similar rates and yields of the
DnaK-mediated reactivation of aggregated G6PDH were obtained when the
DnaK chaperone system, ATP, and pyruvate kinase were added immediately
or after a delay of up to 30 min following heat stabilization (data not shown). This demonstrates the high stability of the G6PDH aggregates. In general, chaperones and ATP were added within 4 min after the final
heat stabilization step of the G6PDH aggregates.
Enzymatic Assays--
The G6PDH activity was measured using the
spectroscopic method as described in Ref. 18. The ATPase activity was
measured by a colorimetric method for phosphate determination using the phosphorus diagnostic reagent from Sigma (19).
Characterization of Inactive G6PDH Species--
To analyze the
mechanism by which the DnaK chaperone system specifically solubilizes
and refolds populations of stable protein aggregates of varying sizes,
we first identified and characterized appropriate substrates for the
reaction. When native G6PDH was pretreated with dithiothreitol and 5 M urea at 47 °C for 5 min and then diluted to final
concentrations ranging from 0.2 to 2.8 µM in folding
buffer, significant levels of spontaneous reactivation occurred at
30 °C. However, spontaneous reactivation was abolished following an
additional incubation of 15 min at 47 °C. Only this complex
procedure (urea and heat denaturation, dilution, and a final heat
stabilization step) was able to reproducibly generate populations of
non-turbid yet stable and inactive aggregates with defined size
distributions (see below). Incubation for 20 min at 65 °C also
yielded stable inactive aggregates that were turbid with undefined high
degrees of complexity.
Gel filtration chromatography of the G6PDH forms that were both
denatured and heat-stabilized at four different concentrations (0.3-1.5 µM, Fig.
1a) showed that the majority
of species was resolved in a continuum of soluble small oligomeric
particles ranging from 106 kDa (Fig. 1a, peak
III) to 5,000 kDa (peak II). Only a minor fraction
eluted in the void volume (5-40 × 103 kDa, according
to the specifications of Amersham Pharmacia Biotech) (Fig.
1a, peak I). As the final concentration at which
the protein was heat-stabilized was increased, the majority of the
protein species in peak II was shifted toward a heavier molecular
weight (Fig. 1a, inset). Although still soluble,
more material eluted in the void volume at the expense of lower
molecular weight forms (Fig. 1a). It should be noted that
because aggregated proteins are likely to assume non-globular shapes,
it is not possible to estimate the precise oligomeric state of the
soluble G6PDH aggregates (4, 5).
The light scattering intensity of the inactive aggregates stabilized at
47 °C was remarkably low; aggregate concentrations of 0.4 and 2 µM scattered light less than 1 and 7%, respectively, when compared with that of the same protein concentrations denatured at
65 °C (Fig. 1b). Moreover, following centrifugation at
16,000 × g for 5 min, the majority of the inactive
G6PDH remained in solution (data not shown). We conclude that in
contrast to the large turbid aggregates formed at 65 °C, the
inactive G6PDH species stabilized at 47 °C are virtually non-turbid.
As demonstrated by gel filtration chromatography, these species were
significantly smaller (consisting of mostly soluble particles) but was
nonetheless as stable as large turbid aggregates and did not refold
spontaneously (data not shown).
Indicative of increased hydrophobic exposure, non-turbid aggregates
bound 7 times more bis-ANS than the native enzyme (Fig. 1c)
and 1.5 times more bis-ANS than the turbid aggregates. This implies
that non-turbid aggregates exposed more hydrophobic residues than
turbid aggregates. bis-ANS binding was proportional to the concentration at which the aggregate was initially stabilized or
subsequently diluted (Fig. 1c).
The Aggregates Are Specific Substrates for the DnaK
System--
All concentrations of the inactivated G6PDH showed less
than 0.1% spontaneous refolding. Moreover, no significant reactivation was observed in the presence of ATP and GroEL/GroES or ClpB or IbpB
chaperones (data not shown). However, in the presence of the DnaK
system and ATP, a variety of aggregate sizes and concentrations were
specifically reactivated after different lag times and at different
rates and yields (Fig. 2a).
This demonstrates that several forms of non-turbid G6PDH aggregates are
authentic substrates that can be specifically disaggregated and
refolded by the DnaK system although in a manner that depends on the
size and concentration of the particles.
GroEL/GroES failed to reactivate even the smallest G6PDH particles
(data not shown) confirming that the size limit for DnaK substrates
exceeds that of GroEL substrates (>60 kDa) (3, 20). However, we also
found that as the size of the aggregates increased, aggregates became
increasingly poorer substrates for the DnaK system. The lag time
necessary to reactivate the same low concentration of 50 nM
G6PDH by the same concentration of DnaK chaperones (3.5 µM) was 24-fold longer with 2.9 µM
aggregated G6PDH than with an 8 times lower concentration of substrate
(0.36 µM) (Fig. 2a, inset). Hence,
unlike a classic enzymatic reaction in which the velocity of the
reaction with a fixed amount of enzyme increases with the concentration
of substrate and becomes maximal in the presence of saturating
concentrations of substrate (Vmax), the reactivation rates carried out by the DnaK system and ATP initially increased up to 0.6 µM G6PDH and then rapidly decreased
as the concentration and size of the aggregates increased above 0.6 µM (Fig. 2b). We found that a 6-fold molar
excess of DnaK over the substrate was necessary to obtain optimal
folding rates and at least a 3-fold molar excess of DnaK for optimal
folding yields. When the DnaK/G6PDH ratio was below 3, the yields
rapidly decreased (Fig. 2b).
DnaK Interacts with Small Protein Aggregates--
In the presence
of increasing concentrations of non-turbid aggregates, the rate of ATP
hydrolysis with a constant concentration of DnaK, DnaJ, and GrpE showed
an activation of up to 2-fold (Fig. 2b). This confirms that
the small G6PDH aggregates are chaperone substrates that interact with
DnaK. The DnaK-ATPase activity initially increased and then decreased
with the substrate concentration. Interestingly, the spectrum of the
DnaK-ATPase activation matched that of the refolding yields rather than
that of the refolding rates (Fig. 2b). This indicates that
the yields of the reaction were affected only when the interaction
between the DnaK system and the aggregated substrate was impaired.
The Refolding Activity of the DnaK System Is Limited by the Size of
the Aggregates--
We next addressed the relationship between the
size of the aggregates and their ability to serve as substrates for the
DnaK system. Small G6PDH aggregates, initially formed and stabilized at
0.4 µM, were concentrated 20-fold and separated by
size-exclusion chromatography (Fig.
3a). Noticeably, the
subsequent gel filtration pattern was virtually identical to that
obtained for the original preparation (data not shown). 87% of the
material was recovered demonstrating that the aggregates were soluble
and highly stable. Three discrete fractions of soluble aggregates
(fractions A, B, and C) were adjusted to the same final concentration
(1 µM) and incubated with the same concentration of the
components of the DnaK system and ATP (as in Fig. 2a).
Remarkably, the DnaK system successfully reactivated some 27% of the
G6PDH in 2 h although only in the fraction that contained the
least complexed aggregates (fraction C). Higher molecular weight
aggregates in fractions B and A remained inactive (Fig. 3b,
striped columns). This confirms that the DnaK system
efficiently refolds only small protein aggregates. Interestingly, a
5-fold dilution to 200 nM of the aggregates allowed the
recovery of some 100 nM G6PDH in fraction C and about half that much in fractions A and B (Fig. 3b, dotted
columns). This suggests that increasing the ratio between the DnaK
system and the substrates can compensate for the decreased ability of
the DnaK system to recognize, disaggregate, and reactivate large
protein aggregates.
The Chaperone/Substrate Ratio Determines the Refolding Efficiency
of Large Aggregates--
We specifically addressed the role of the
ratio between DnaK and large aggregates on the efficiency of the
chaperone system. The refolding activity was measured with two types of
G6PDH aggregates: small aggregates generated at 0.4 µM
that were subsequently concentrated to 2 µM or large
non-turbid aggregates that were directly generated at 2 µM. The yield of chaperone-mediated reactivation of small aggregates (formed at 0.4 µM and concentrated thereafter
to 1 µM) was 2 times higher than with the same amount (1 µM) of large aggregates formed at 2 µM and diluted thereafter (Fig.
4a). However, the same
concentration of chaperone became equally efficient at reactivating a
5-fold smaller amount (0.2 µM) of aggregated G6PDH regardless of the particle size (Fig. 4b).
Gel filtration confirmed that neither concentrated small aggregates
(0.4 µM) nor diluted large aggregates (2 µM) changed oligomeric state during concentration or
dilution (Fig. 4, a and b, insets) indicating that both oligomeric structure and the concentration of the
aggregate determine the efficiency of the chaperone system. The
dependence of the chaperone efficiency on the size and concentration of
the aggregate was further addressed over a wide range of concentrations (0.05-2.0 µM) of the small and large types of
aggregates described above (Fig. 4c). At concentrations
up to 0.6 µM G6PDH (a DnaK/G6PDH ratio of >6), the
reaction yield was independent of the oligomeric state of the
aggregate, but when the substrate concentration exceeded 0.6 µM (a DnaK/G6PDH of <6) the reaction yields strongly
decreased. The decrease of the chaperone efficiency was more dramatic
with large aggregates than with small aggregates. At 2 µM
substrate, the reactivation yields of the reaction with large
aggregates was 4 times lower than with small aggregates. This suggests
that productive refolding requires concomitant interactions of several chaperone molecules with the same polypeptide within a large aggregate.
Higher DnaK Concentrations Compensate for Reduced Refolding
Activity--
We further addressed the possibility that limiting DnaK
concentrations account for the observed low chaperone efficiency with high aggregate concentrations. The low refolding rates and yields of
the reaction of large aggregates (2 µM) were dramatically
improved when the DnaK concentration was increased by a factor of 3 from 3.5 to 10 µM (the DnaJ and GrpE remained unchanged).
A similar effect was observed when 6.5 µM DnaK was
supplemented 150 min after initiation of poorly productive
refolding to the initial 3.5 µM DnaK (Fig.
5a). This indicates that the
ratio between DnaK and the substrate determines the effectiveness of
the interaction of the DnaK system with large aggregates. Refolding was
similarly improved when the concentration of all three components of
the DnaK system was proportionally increased (see below) indicating that the effect primarily depends on the DnaK concentration relative to
the substrate rather than on the ratio between DnaK and its co-chaperones, DnaJ and GrpE.
ClpB Can Substitute for High DnaK Concentrations--
When
inefficient refolding of high molecular weight aggregates (2 µM) with 3.5 µM DnaK (and 0.7 µM DnaJ and 0.35 µM GrpE) was tested in the
presence of a substoichiometric level of the ClpB chaperone (0.5 µM), both rates and yields of reactivation were
dramatically increased (Fig. 5b). The effect of 0.5 µM ClpB concentrations was similar to that of 10 µM DnaK.
In contrast to non-turbid G6PDH particles, a low concentration of large
turbid particles (0.4 µM) formed at 65 °C was not reactivated by 3.5 µM DnaK and was only poorly
reactivated by 10 µM DnaK (Fig. 5c). Only in
the presence of 0.5 µM ClpB and 3.5 µM DnaK
(and 0.7 µM DnaJ and 0.35 µM GrpE) were the
turbid particles reactivated 8 times faster, reaching yields 7 times higher than in the presence of 10 µM DnaK without ClpB.
Thus, ClpB appears to render the large turbid aggregates much more
compatible for subsequent disaggregation by the DnaK system. Moreover,
ClpB significantly reduced the amount of DnaK needed for the
disaggregation of non-turbid aggregates and was absolutely essential
for the disaggregation of large turbid aggregates.
We tested the ability of 3.5 µM DnaK (and its
co-chaperones) to refold increasing concentrations and sizes of
aggregate particles with or without ClpB (Fig. 5d). The
presence of ClpB nearly doubled the recovery of the small aggregates.
Remarkably, the ClpB was most effective at alleviating ineffective
refolding of large non-turbid aggregates at concentrations of up to 2.9 µM by the DnaK system alone. In the case of the large
aggregates, the refolding yields were 28-fold higher than without ClpB
(Fig. 5d, inset). This demonstrates that in the
presence of substoichiometric amounts of ClpB, the yields of
DnaK-mediated disaggregation and reactivation become equally optimal
with all forms of aggregates from small non-turbid to infinitely large
turbid aggregates. Thus, confirming initial observations on large
aggregates of malate dehydrogenase (12), ClpB alleviates the dependence
of the DnaK chaperone machinery on the size and of the aggregated substrates.
The effect of ClpB was confirmed when the refolding yields were
analyzed in the presence of increasing concentrations of DnaK (and
proportionally of DnaJ and GrpE), without or with ClpB (Fig. 5e). Without ClpB, half of the maximal refolding was reached
in the presence of about a 4-fold molar excess of DnaK over G6PDH, whereas a near equimolar amount of DnaK sufficed in the presence of
ClpB. This can be interpreted as if ClpB increased the apparent affinity of DnaK for the aggregates by increasing the ability of DnaK
molecules to interact with the aggregates.
During the denaturation of a native protein, unstable folding
intermediates that escape chaperone binding may reach an alternatively stable state of inactive insoluble aggregates (5). Stable aggregates remain refractory to subsequent attempts of in vitro
reactivation by most individual chaperone systems such as GroELS or
DnaK-DnaJ-GrpE (8, 9, 10, 17, 20). Only in the specific case of
heat-inactivated RNA polymerase was the DnaK chaperone system from
E. coli and ATP able to mediate efficient disaggregation and
reactivation, an exceptional activity that was attributed to an
undefined "mild" nature of the aggregate (6, 7).
To better characterize the nature of the protein aggregates that can
effectively serve as chaperone substrates and be specifically solubilized by the DnaK system, we developed a method to generate various populations of stable aggregates of G6PDH with a controlled spectrum of particle sizes. These range from small soluble non-turbid aggregates to large yet mostly soluble non-turbid aggregates to even
larger and insoluble turbid aggregates. The G6PDH aggregates were
stable both in their oligomeric structure and in their ability to serve
as chaperone substrates. They did not refold spontaneously. Size-exclusion chromatography indicated a complexity that was unaffected by dilution or concentration. Similar refolding efficiencies were obtained when chaperones were added immediately or following hours
of delay. bis-ANS binding was strictly proportional to the concentration of the protein. Hence, the general hydrophobic exposure of the aggregate surfaces was unaffected by the complexity and concentration of the aggregate. Because increasingly larger globular particles display decreasing surface/volume ratios, a constant ratio
suggests that G6PDH aggregates are mostly elongated filaments typical
of many protein aggregates (4, 5).
Remarkably, the large turbid aggregates formed at 65 °C exposed
fewer hydrophobic residues, thus suggesting more collapsed and compact
structures. Because hydrophobic residues are core elements of the DnaK
and DnaJ binding sites (21), we expected that fewer chaperone molecules
would be able to interact with insoluble turbid aggregates than with
soluble non-turbid aggregates. This was confirmed by substrate-induced
activation of the DnaK ATPase. Whereas DnaK strongly interacted with
small non-turbid aggregates, interaction decreased with large
non-turbid aggregates and was negligible with very large turbid
aggregates (data not shown).
The interaction of the DnaK system with small non-turbid aggregates was
highly productive in terms of protein reactivation. This implies that
unlike GroELS chaperonins, the DnaK system can bind locally and
initiate assisted refolding in a local region of a polypeptide while
still being involved in strong protein-protein interactions within the
aggregate. Noticeably, protein reactivation by the DnaK system became
increasingly futile as the complexity of the aggregate increased and
the hydrophobic exposure decreased. This can be attributed to the
reduced exposure of chaperone binding sites in large non-turbid
aggregates and in turbid aggregates when compared with small soluble
aggregates. Consistent with this interpretation is our finding that at
the same concentration of aggregates (1 µM) only the
fraction containing the lowest mass (not the medium or large soluble
aggregates isolated by gel filtration chromatography) was efficiently
refolded by the DnaK system. On the other hand, although dilution did
not affect the complexity of large aggregates, the yields of refolding
were about 50 times higher with 0.2 µM than with 1 µM of the same large aggregates (Fig. 3, fractions A and
B). Thus, a higher ratio of DnaK per G6PDH can compensate for the
decreased efficiency of reactivation of large aggregates. Indeed, large
aggregates were efficiently reactivated when the DnaK concentration was
increased to a 5-fold excess (10 µM) over the substrate.
A non-physiological increase of the concentration of only DnaJ or GrpE
resulted in a strong inhibition of the chaperone activity with all
types of aggregates (data not shown). However, as with DnaK alone, the
inhibited reactivation was similarly alleviated by the concomitant
increase in DnaK, DnaJ, and GrpE levels while keeping a fixed ratio of
10:2:1 between the respective members of the chaperone system (Fig.
5e).
Moreover, the dose response of the DnaK system indicates that in the
absence of ClpB, the mechanism of productive disaggregation requires
some sort of a cooperative behavior between different DnaK molecules
because efficient refolding requires that more than one chaperone
molecule interacts at the same time with the same polypeptide within
the aggregate. Because the algorithm of Rüdiger et al.
(21) predicts as many as 12 putative high affinity DnaK-binding sites,
several DnaK molecules can theoretically bind to the same G6PDH
polypeptide. Our data, however, suggest that as the aggregates became
larger and more turbid, more sites seemingly became buried within the
structure and were less available for DnaK binding.
A combination of the yeast chaperones hsp104, hsp70, and hsp40
initially demonstrated some disaggregation activity in vitro and solubilization of pre-aggregated proteins (9). The hsp104 homologue, ClpB, was further demonstrated to interact directly with
large turbid protein aggregates, and together with ATP it induced
structural changes in the aggregates. Hydrophobic regions became
transiently exposed and available for subsequent interactions with the
DnaK system leading to efficient disaggregation activity (12). We
demonstrate in this study that for efficient solubilization of large
aggregates the presence of ClpB reduces the need for an excess of DnaK.
A dose response of the DnaK system in the presence of a constant
concentration of substrate suggests that efficient reactivation with
ClpB requires 4-5 times less DnaK per substrate than reactivation
without ClpB.
There is no obvious Hsp100 homologue in the complete genome of
Drosophila melanogaster. Our finding that protein
disaggregation can take place with large amounts of Hsp70 even without
Hsp100 correlates well with the exceptionally high levels of Hsp70
found in Drosophila under stress. Interestingly, Hsp70
overexpression suppresses polyglutamine aggregate-mediated
neurodegeneration in Drosophila (31).
The Mechanism of DnaK-mediated Disaggregation--
In the presence
of ClpB, the mechanism of productive disaggregation exempts the DnaK
molecules from having to act in an apparent cooperative manner (Fig.
5e). We suggest a two-step cooperative mechanism for the
DnaK-DnaJ-GrpE-mediated solubilization and reactivation of stable
protein aggregates. During the first step, several DnaK molecules must
initially interact with exposed hydrophobic regions preferentially in
the same aggregated polypeptide and thus prevent intramolecular
collapses and intermolecular re-aggregation. During the second step, a
dynamic binding and release of DnaK in the presence of DnaJ, GrpE, and
ATP allows folding of previously bound regions, whereas the rest of the
molecule is maintained in a partially non-collapsed state by other
bound DnaK molecules. This would explain the observed apparent
cooperative effect between DnaK molecules, which was canceled in the
presence of ClpB. In Ref. 12, we proposed a mechanism in which ClpB
hexamers shear ClpB-bound aggregates and thus actively expose some of
the hidden hydrophobic regions on the aggregates. Hence, although
through a different mechanism, ClpB could maintain aggregated
polypeptides in a partially non-collapsed state and thus functionally
surrogate the holding role otherwise carried out by excess DnaK. ClpB
activity would thus free the excess DnaK molecules to instead perform
repetitive cycles of productive assisted refolding (with DnaJ and GrpE)
on local exposed regions in the aggregated polypeptide, leading to efficient disaggregation and renaturation.
Implications for the Mechanism of DnaK and for the Chaperone
Network in the Cell--
Together with the evidence that the DnaK
system alone can bind and initiate the folding of polypeptides that are
still aggregated, the efficient folding of large aggregates by near
stoichiometric amounts of DnaK in the presence of ClpB strongly
suggests that the mechanism of DnaK involves repetitive cycles of local
rather than global assisted folding.
In the cell, the efficient rescue of thermolabile proteins during and
after stress depends on the nature of the unfolded intermediates generated by each protein and on the availability and specificity of
the various components of the chaperone network for unfolding intermediates. Thus, GroEL may preferentially interact with partially denatured molten globules (22), IbpB with extensively unfolded proteins
(17), and ClpB with large turbid aggregates (12). In contrast, the DnaK
system may interact with small segments of about 8-9 residues in
extensively unfolded proteins (23) as well as in exposed polypeptide
loops within large yet non-turbid protein aggregates (this work).
Because the DnaK system interacts with small extended segments, this
chaperone system has been initially assigned to an early stage in the
sequential folding pathway of nascent and newly translocated proteins
prior to GroEL/GroES chaperonins (24). However, other studies have
shown that the DnaK system can efficiently process folding
intermediates handled from small heat shock proteins (17), components
of the import complex of organelles (25, 26), and from GroEL (27). In
addition, the DnaK system functionally cooperates with trigger factor
(28), Hsp90 (29), and ClpB (12, 13). Our finding that small aggregates
can be actively solubilized by the DnaK machinery has a major
consequence for the role of the DnaK system within the chaperone
network of the cell. It can (i) receive and process soluble
intermediates from import pores, ribosomes, and other chaperones, (ii)
resolubilize early forms of aggregates that accidentally form as
by-products of the folding activity of other chaperones such as
GroEL/GroES (30), and (iii) disaggregate small size aggregates
generated during stress. Thus the hsp70 system can reintroduce
polypeptides trapped in small aggregates back into the chaperone
network for refolding or into the protease network for degradation.
We thank H-J. Schönfeld for purified
DnaK, DnaJ, and GrpE, C. Squires for plasmid pClpB, and S. Rüdiger for predicting the number of DnaK-binding sites in
G6PDH.
*
This work was supported in part by grants from the German
Israeli Foundation and the Abisch-Frenkel Foundation (to P. G.) and
from the Deutsche Forschungsgemeinschaft (to B. B.).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.
¶
To whom correspondence should be addressed. Tel.:
972-2-6585391; Fax: 972-2-6584425; E-mail:
pierre@vms.huji.ac.il.
Published, JBC Papers in Press, May 2, 2000, DOI 10.1074/jbc.M001293200
The abbreviations used are:
G6PDH, glucose-6-phosphate dehydrogenase;
bis-ANS, 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid.
Size-dependent Disaggregation of Stable Protein
Aggregates by the DnaK Chaperone Machinery*
,,¶
Silberman Institute of Life Sciences, The
Hebrew University of Jerusalem, 91904 Jerusalem, Israel and the
§ Institut für Biochemie und Molekularbiologie,
Universität Freiburg, Hermann-Herder Strasse 7, D-79104
Freiburg, Germany
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-pleated structures and exposed hydrophobic surfaces
that remain inactive after stress (4, 5).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (34K):
[in a new window]
Fig. 1.
Characterization of denatured G6PDH.
a, size fractionation of denatured G6PDH aggregates. G6PDH
was urea/heat-denatured at 0.3 µM (filled
circles), 0.6 µM (open squares), 1.2 µM (filled diamonds), and 1.5 µM
(open triangles) as described under "Materials and
Methods" and fractionated by gel filtration chromatography.
Peak I, aggregates eluting in the void volume above 5,000 kDa; peak II, aggregates below 5,000 kDa but above 106 kDa;
peak III, inactive 106-kDa G6PDH dimers; inset,
estimated molecular mass of the most abundant species in peak II
(arrowheads) as a function of the concentration at which the
G6PDH aggregates were stabilized. b, turbidity of denatured
G6PDH. Light scattering intensity at 550 nm of urea/heat-denatured
G6PDH at increasing concentrations (filled circles) was
compared with heat-denatured G6PDH (65 °C) at 2.5 µM
and diluted to the indicated concentrations (filled
triangles). c, hydrophobic exposure of denatured G6PDH.
The fluorescence intensity of bis-ANS in the presence of
urea/heat-denatured G6PDH at increasing concentrations (filled
circles) was compared to G6PDH that was urea/heat-denatured at 2.0 µM and then diluted to the indicated concentrations
(open triangles), to native G6PDH (open circles),
or to G6PDH that was heat-denatured at 65 °C at 2.0 µM
and diluted to the indicated concentrations (filled
triangles).
View larger version (30K):
[in a new window]
Fig. 2.
Effect of the aggregate concentration on DnaK
chaperone-mediated refolding. The reactivation of preformed
aggregates, urea and heat-denatured at the indicated concentrations,
was performed in the presence of a fixed amount of DnaK, DnaJ, and GrpE
at 3.5, 0.7, and 0.35 µM, respectively, and 3 mM ATP in the presence of an ATP-regenerating system, as
described under "Materials and Methods." a,
time-dependent reactivation of urea/heat-denatured G6PDH at
the indicated concentrations. Inset, time needed to
reactivate 50 nM G6PDH from different aggregate
concentrations. b, refolding rates (filled
circles), absolute refolding yields (open triangles) of
active G6PDH, and rates of DnaK-ATPase (open squares) as a
function of substrate concentration.
View larger version (25K):
[in a new window]
Fig. 3.
Effect of size of aggregates on Dnak-mediated
refolding. a, gel filtration profile,
urea/heat-denatured G6PDH aggregates were formed and heat-stabilized at
0.4 µM (as in Fig. 1a) and then concentrated
50-fold and separated by size-exclusion chromatography. Three fractions
of soluble aggregates were collected (A, 8.5-11 ml;
B, 11-13.5 ml; and C, 14-16.5 ml).
b, refolding activity. Fractions A, B,
and C were adjusted to a final concentration of 1.0 µM (striped column) or 0.2 µM
(dotted column) and incubated 2 h with DnaK, DnaJ, and
GrpE at 3.5, 0.7, and 0.35 µM, respectively, and 3 mM ATP (as described in the Fig. 2 legend).
View larger version (21K):
[in a new window]
Fig. 4.
Effect of particle concentration and size on
DnaK-mediated refolding. a, reactivation of 1.0 µM small particles initially prepared at 0.4 µM and concentrated thereafter (open circles)
or 1.0 µM large aggregates initially prepared at 2 µM and diluted twice thereafter (filled
circles) with DnaK, DnaJ, and GrpE (3.5, 0.7, and 0.35 µM, respectively). Upper inset, gel filtration
elution profile of the 0.4 to >2 µM sample after
concentration; lower inset, elution profile of the sample
initially prepared at 2 µM. b, reactivation by
the DnaK system of 0.2 µM small particles (open
circles) or 0.2 µM large particles (filled
circles) as shown in a. Upper inset, the
elution profile of the sample initially formed at 0.4 µM;
lower inset, the elution profile of 5× dilution of the
sample initially stabilized at 2 µM (2 to >0.4
µM). Elution profiles were expressed in percent of the
maximal signal. c, reactivation by DnaK, DnaJ, and GrpE
(3.5, 0.7, and 0.35 µM, respectively) of various
concentrations (0.05-2 µM) of small (open
circles) or large (filled circles) aggregates as shown
in a.
View larger version (19K):
[in a new window]
Fig. 5.
High DnaK or low ClpB concentrations activate
DnaK-mediated refolding of large aggregates. a, effect
of DnaK concentrations. 2 µM non-turbid aggregates were
reactivated in the presence of constant 0.7 µM DnaJ, 0.35 µM GrpE, and 3.5 µM DnaK (filled
circles) or 10 µM DnaK added at time 0 (filled
triangles) or 6.5 µM DnaK supple- mented after 150 min of reaction (arrow) with low DnaK
(open triangles). b, reactivation of 2 µM aggregates by DnaK, DnaJ, and GrpE (KJE)
(3.5, 0.7, and 0.35 µM, respectively) without
(filled circles) or with 0.5 µM ClpB
(open circles). c, reactivation of 0.4 µM turbid aggregates (formed at 65 °C), by DnaK, DnaJ,
and GrpE alone (filled circles, KJE) or supplemented after
40 min with 6.5 µM DnaK (arrowhead, filled
triangles) or with 0.5 µM ClpB as in a
(open circles). d, effect of concentration and
size of G6PDH aggregates on the yields of chaperone-mediated
reactivation by DnaK, DnaJ, and GrpE (3.5, 0.7, and 0.35 µM, respectively), without (filled circles,
KJE) or with 0.5 µM ClpB (open circles,
KJE+C1pB). Inset, activation effect of ClpB on
DnaK-mediated disaggregation of increasingly large and abundant
aggregates. e, dose-response curve of DnaK, DnaJ, and GrpE
concentrations (constant molar ratio of 10:2:1 between co-chaperones)
as a function of the yields of G6PDH reactivation from non-turbid large
aggregates formed at 1.8 µM without (filled
circles) or with 0.5 µM ClpB (open
circles).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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