From the Department of Plant Sciences, Silberman
Institute of Life Sciences, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel and the § Institut für Biophysik and
Physikalische Biochemie, Universität Regensburg, Postfach,
93040 Regensburg, Germany
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ABSTRACT |
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The role of small heat-shock proteins in Escherichia coli is still enigmatic. We show here that the small heat-shock protein IbpB is a molecular chaperone that assists the refolding of denatured proteins in the presence of other chaperones. IbpB oligomers bind and stabilize heat-denatured malate dehydrogenase (MDH) and urea-denatured lactate dehydrogenase and thus prevent the irreversible aggregation of these proteins during stress. While IbpB-stabilized proteins alone do not refold spontaneously, they are specifically delivered to the DnaK/DnaJ/GrpE (KJE) chaperone system where they refold in a strict ATPase-dependent manner. Although GroEL/GroES (LS) chaperonins do not interact directly with IbpB-released proteins, LS accelerate the rate of KJE-mediated refolding of IbpB-released MDH, and to a lesser extent lactate dehydrogenase, by rapidly processing KJE-released early intermediates. Kinetic and gel-filtration analysis showed that denatured MDH preferentially transfers from IbpB to KJE, then from KJE to LS, and then forms a active enzyme. IbpB thus stabilizes aggregation-prone folding intermediates during stress and, as an integral part of a cooperative multichaperone network, is involved in the active refolding of stress-denatured proteins.
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INTRODUCTION |
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The small heat-shock proteins (sHSPs)1 belong to a ubiquitous family of low molecular mass (15-30 kDa), stress-induced proteins in prokaryotes and eukaryotes. Whereas various sHSPs share weak sequence homologies (1, 2), many sHSPs appear to be functionally and structurally related. Many sHSPs assemble into large globular complexes, whose oligomeric structures may vary depending on the degree of subunit phosphorylation or the concentration of ions (3-5). The overexpression of sHSPs in plant, yeast, and in mammal cells correlates with increased levels of thermal resistance (6-9). In vitro, sHSPs specifically recognize, bind, and prevent the aggregation of non-native proteins during stress (3-5, 10), suggesting that similarly to GroEL/GroES (LS) and Hsp70 (11, 12), sHSPs can serve as an efficient binding reservoir for unstable protein-folding intermediates during stress. However, at variance with other molecular chaperones, such as Hsp70, Hsp60, Hsp104, and Hsp90 (for a review, see Ref. 13), small HSPs do not hydrolyze ATP and do not display a specific ability to promote the correct refolding of the bound stabilized proteins (3-5).
The small heat shock proteins IbpA and IbpB from Escherichia
coli are two sequence-related 14- and 16-kDa proteins,
respectively, co-transcribed during stress by the bacterial heat-shock
transcription factor 32 (14). IbpA and IbpB share a low
sequence homology in their C-terminal region with sHSPs from yeast,
plants, and mammals, including
B-crystallines (14-19, 2).
Furthermore, they seem to be distantly related to other bacterial
chaperones such as PapD and Caf1M. Based on the resolved x-ray
structure of the PapD chaperone and on sequence homology, a model has
been proposed where the three-dimensional structure of IbpB resembles
that of immunoglobulins (20).
In E. coli, IbpA and IbpB are found associated with endogenous proteins that aggregate intracellularly during heat-shock (21) and with non-native recombinant proteins in inclusion bodies (14). They are implicated in the solubilization of protein aggregates after stress (21). In vitro, IbpB is a large globular complex, whose oligomeric state may vary at different temperatures and ionic concentrations.2 Similarly to other small HSPs, light-scattering measurements indicate that IbpB can specifically bind thermally or chemically denatured proteins such as MDH, LDH, and citrate synthase and thus prevent their irreversible aggregation (not shown).
We show here that IbpB can bind and stabilize denatured proteins, and furthermore, deliver them to the DnaK/DnaJ/GrpE (KJE) chaperone system for subsequent active ATP-dependent refolding. KJE-mediated refolding of IbpB-stabilized proteins can be further activated by LS chaperonins, demonstrating cooperation between the different chaperone systems. IbpB can therefore function as a primordial protein-binding element of a multichaperone network, involved in stabilizing and active refolding of stress-denatured proteins.
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EXPERIMENTAL PROCEDURES |
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Phage Complementation-- Wild type and E. coli B mutant T850 (22) were used for in vivo complementation assays of T4 morphogenesis as described in Ref. 23. Plasmids pBF3-IbpA and pBF3-IbpB were from Dr. C. Hergersberg, Boehringer Mannheim GmbH, and pSE420 was from Invitrogen Inc.
Proteins--
IbpB was purified as follows: late log phase
E. coli cells, containing plasmid pBF3-IbpB in LB medium and
chloramphenicol (50 µg/ml), were incubated for an additional 12 h at 37 °C in the presence of
isopropyl-1-thio--D-galactopyranoside (100 µg/ml), collected, and resuspended in 50 mM Tris-HCl, pH 7.5, 150 mM KCl, 20 mM MgAc2 (Buffer A),
containing 5 µg/ml leupeptin. Cells were disrupted five times at
25 °C in a French Press at 900 p.s.i. Soluble proteins in the
30,000 × g supernatant were incubated with 6%
polyethylene glycol 6000 (Merck) for 30 min at 4 °C. The protein
pellet from 30,000 × g was resuspended in buffer A,
filtered through a 0.45-µm filter (Schleicher & Schuell), and
separated by gel filtration (0.5 ml/min) on a semi-preparative Superose 6B column (Pharmacia) in buffer A. The fractions collected between 6.0 and 7.5 ml (above 2.106 daltons), which were highly
enriched with soluble oligomeric IbpB, were applied to a resource-Q
column (Pharmacia), equilibrated with buffer A. Elution was carried out
with a linear gradient from 150 to 500 mM KCl in Buffer A. The fractions eluting at 230 ± 30 mM were collected
and frozen as 34-45 µM (protomer) 95% pure IbpB stock
solutions.
3H Labeling-- Native MDH was labeled with NaB(3H)4 as described in Ref. 11. After purification by gel filtration (Superose 6B column, Pharmacia), 3H-labeled MDH was found to be as active as unlabeled MDH. During heat-denaturation and chaperone-assisted refolding, 3H-labeled MDH behaved indistinguishably from unlabeled MDH (not shown).
Chaperone Activity Assays-- MDH was heat-denatured for 30 min at 47 °C as in Ref. 11 in the presence of various amounts of IbpB, DnaK, DnaJ, and/or GroEL, GroES as specified and subsequently refolded at 25 °C in the presence of supplemented DnaK/DnaJ/GrpE and/or GroEL/GroES (4, 4, 1, 4, and 6 µM, respectively) in 50 mM triethanolamine, pH 7.5, 20 mM MgAc2, 150 mM KCl, 5 mM dithiothreitol, 3 mM phosphoenol pyruvate, 7 µg/ml pyruvate kinase (Sigma), and 2 mM ATP (buffered with KOH), as indicated.
Enzymatic Assays-- The activity of MDH was measured as described in Ref. 27, at 25 °C in 150 mM potassium phosphate buffer, pH 7.5, 10 mM dithiothreitol, 0.5 mM oxaloacetate, and 0.28 mM NADH (Sigma). The activity of LDH was measured as described in Ref. 28 in 100 mM Tris, pH 7.5, 0.5 mM pyruvate, and 0.28 mM NADH. The time-dependent oxidation of NADH by MDH or LDH was monitored at 340 nm. Native MDH dimers remain stable and active in solutions above a concentration of 6 nM (protomers) (29). The apparent rates of protein refolding were calculated from the time-dependent changes in the enzymatic activity of chaperonin solutions containing more than 20 nM enzyme.
Gel Filtration--
3H-Labeled MDH (0.25 µM) was heat-denatured in the presence of IbpB (4 µM), DnaK + DnaJ (4, 0.8 µM) or GroEL (4 µM), and incubated for 1 h at 25 °C with ATP and
ATP-regeneration systems, in the absence or presence of KJ, KJEL, or L
(Fig. 5). A Superose 6B gel filtration column (Pharmacia) (at 0.5 ml
min1), run in the presence of the refolding buffer and 1 mM ATP was used for separation. 3H-Labeled
samples were collected at the indicated elution volumes (Fig. 5), mixed
with a 6-fold volume of LumaxTM, and counted.
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RESULTS |
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IbpA and IbpB Can Suppress a Phage Growth groEL Mutation in E. coli-- Wild type E. coli B cells are highly sensitive to T4 phages (23). In contrast, the mutant strain T850, which contains a chromosomal groEL with a R268C mutation,3 do not support T4 growth (Fig. 1). We found that phage growth was specifically restored in T850 cells containing plasmid-encoded ibpA or ibpB genes (Fig. 1), however, only in the presence of a higher phage titer (108 particles/ml) than in wild type cells.
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IbpB-stabilized MDH Can be Specifically Refolded by KJE + ATP--
When exposed to 47 °C, the thermolabile enzyme MDH is
inactivated at an apparent rate of 0.13 min1 (11) and
forms large insoluble protein aggregates that do not refold
spontaneously after the heat shock (not shown). In contrast, when
exposed to heat stress in the presence of a 4-fold molar excess of
thermostable IbpB, MDH was inactivated at a slower rate (0.10 min
1) and formed a stable soluble IbpB·MDH
complex.2 We found that the IbpB·MDH did not
significantly reactivate, even after a 5-h incubation at room
temperature (Fig. 2A).
However, when IbpB·MDH was supplemented with KJE chaperones and ATP,
a significant amount (10%) of native MDH was slowly recovered, at a
slow rate of ~0.7 nM min
1. This
KJE-mediated refolding of IbpB·MDH was a specific reaction, as it did
not occur when IbpB was absent during the heat denaturation (Fig. 2,
A and B), or when ATP or GrpE were absent during
renaturation (not shown).
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KJE-mediated Refolding of IbpB·MDH Is Activated by LS--
When
IbpB·MDH was supplemented with LS chaperonins and ATP, only
insignificant amounts of MDH were reactivated (1.5%), in addition to
the small amounts generated by spontaneous refolding (2.5% within 4-5
h, Fig. 2A). Remarkably, when KJE and LS chaperones and ATP
were concomitantly added to IbpB·MDH after the heat shock, a major
fraction (40%) of the MDH was actively recovered at an apparent rate
5.5 times higher (3.7 nM min1), than without
LS (Fig. 2A). Interestingly, a significant amount (10%) of
MDH that was heat-inactivated without IbpB was also recovered at a slow
rate of 0.8 nM min
1, but only in the presence
of the two chaperone systems KJE and LS (Fig. 2B). The
strong dependence of the refolding rates and yields of MDH, on the
presence of IbpB during denaturation, could be used as a first
functional assay for a small heat-shock protein under stringent
conditions.
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Gel Filtration Analysis of Sequential MDH Transfer from IbpB to KJE and LS-- Gel filtration of chaperone complexes with bound heat-denatured 3H-labeled MDH provided direct evidence for directional MDH transfer from IbpB to KJE, then to LS, during reactivation (Fig. 5). While native complexes of IbpB exceed 2 × 106 daltons2 and consequently eluted near the void volume of the column (6-7.5 ml), individual native complexes of GroEL, KJ, or MDH, independently resolved at 11-13, 13.5-15.5, and 15.5-18 ml, respectively (arrows shown instead of profiles, Fig. 5). The elution profile of [3H]MDH, which had been heat-denatured in the presence of IbpB alone and then incubated 1 h with ATP alone prior to injection, distributed in two peaks; one together with IbpB, and the other in the low molecular weight region corresponding to free MDH (Fig. 5, profile 1). The elution profile of heat-denatured IbpB-bound MDH, incubated 1 h with KJ and ATP but without GrpE, showed 3H label associated to the broad KJ fraction, at the expense of the IbpB containing and the free MDH fractions (Fig. 5, profile 2). Thus, a significant amount of the non-native MDH was transferred after the heat shock from IbpB and from the free state to the KJ chaperones. In contrast, when IbpB-MDH was incubated with ATP, KJ, and in addition with E + L (without S), the majority of the 3H label eluted associated to the GroEL fraction, at the expense of the IbpB-, KJ-, and free MDH-containing fractions (Fig. 5, profile 3). Thus, a significant amount of IbpB bound and free MDH, that was initially transferred to KJ, was further transferred to the GroEL.
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DISCUSSION |
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Whereas small Hsps share with other molecular chaperones the ability to recognize, bind, and prevent the aggregation of non-native proteins under stress, the classification of small Hsps as true chaperones remains an open question because of their poor performance at specifically promoting the correct refolding of the sHsps-stabilized proteins. A first indication that sHsps may collaborate with other chaperones in the refolding of bound denatured proteins came from the observation that the refolding of heat-denatured citrate synthase bound to mammalian Hsp25, can be activated by Hsp70, even without ATP (4).
We observed here that similarly to other small Hsps from eukaryotes
(3-5, 10, 16), IbpB from E. coli can stabilize denatured proteins such as citrate synthase, -glucosidase, and MDH in a soluble non-aggregated form.2 Remarkably, whereas IbpB
alone did not promote the active refolding of bound proteins after the
stress, IbpB-bound MDH or IbpB-LDH were specifically reactivated by KJE
chaperones and ATP, but not directly by LS chaperonins. Hence, the
apparent rate of refolding of IbpB-MDH was 50 times higher in the
presence of KJE + LS chaperones and ATP, then without KJE + LS and the
yield of the reaction was about one recovered MDH for 10 IbpB subunits
present during denaturation. Similarly, ATP- and KJE-assisted refolding
of IbpB-LDH was 70-90 times faster than without ATP or KJE.
The kinetic and gel filtration data strongly suggested that MDH, but
not necessarily LDH or glucose-6-phosphate dehydrogenase, was refolded
in a sequential, multichaperone reaction. When native MDH was heat
denatured in the presence of IbpB alone, an IbpB·MDH complex was
formed which was in equilibrium after the heat shock with a free
inactive MDH species. The IbpB-released MDH tended to convert into a
kinetically trapped inactive species, unless allowed to bind KJE, but
not LS chaperones. The slow dissociation of MDH from the IbpB·MDH
complex was the rate-limiting step. Delays in the addition of the
various chaperone components showed that MDH was first transferred to
KJE chaperones, where it accumulated, unless allowed to partially fold
and then interact with LS chaperonins. In contrast with the
IbpB-released species (MDH1), which was characterized by a
high affinity for the KJE chaperones and a low (non-productive)
affinity for LS chaperonins, the KJE-released species (MDH2)
had a low affinity for IbpB and a high affinity for LS chaperonins. The
increased affinity of IbpB-released MDH1 for KJE, and of
KJE-released MDH2 for LS provides a preferential pathway for
the sequential MDH refolding in the multichaperone network, as follows:
IbpB-MDH1 + KJE + LS IbpB + KJE-MDH1 + LS
IbpB + KJE + LS-MDH2
IbpB + KJE + LS + MDHnat.
Binding to IbpB during the heat shock was also a prerequisite for a strict KJE- and ATP-dependent refolding of urea/heat-denatured LDH. However, KJE-released LDH species proceeded to the native state almost as rapidly without as with LS chaperonins, suggesting that LS was optional. Interestingly, glucose-6-phosphate dehydrogenase displayed a yet different behavior. Fluorescence and light scattering indicated that heat-inactivated glucose-6-phosphate dehydrogenase was neither aggregated nor bound to IbpB or LS. Whereas denatured glucose-6-phosphate dehydrogenase did not refold spontaneously, it was specifically refolded by KJE + ATP, but not by IbpB and/or by LS + ATP (data not shown). Hence, IbpB can serve as a specific high capacity scavenger for aggregation-prone folding intermediates, but it does not necessarily interact with all types of denatured proteins.
It should be noted that the sequential IbpB KJE
LS mediated
refolding of MDH was observed only when MDH was heat denatured in the
presence of IbpB alone. When saturative amounts of LS and KJE were also
present during the denaturation, IbpB displayed the lowest affinity for
denatured MDH. This implies that in the cell, various protein
intermediates can bind and distribute according to the relative
amounts, binding capacities of each of the chaperones present, and
according to the specific affinity of the folding intermediates for the
chaperones present during the stress.
We propose a model that describes the various protein-folding pathways
that may exist in the multichaperon network (Fig. 6). Upon
denaturation, native proteins form an unstable folding intermediate that can either aggregate irreversibly, refold spontaneously
(pathway 1), or distribute
between several components of the chaperone network. Whereas
acid-denatured barnase refolds spontaneously without chaperones
(31), heat-denatured MDH can bind all three components, depending
on their relative abundance during denaturation. IbpB-MDH sequentially
transfers to KJE, then to LS (pathway 4 5
6).
KJE-MDH and LS-MDH preferentially used pathways 3
6 and
2, respectively. Whereas, LDH denatured in urea for 30 s is preferentially refolded by LS (pathway 2) (11),
urea-heat denatured LDH was refolded by IbpB and KJE, but not by LS
(pathway 4
5). In contrast, the refolding of firefly
luciferase denatured by guanidinium chloride and heat first required
LS, then KJE (pathway 2
6) (32). In a yet different
pathway, heat-denatured glucose-6-phosphate dehydrogenase was refolded
by KJE and not by IbpB or LS (pathway 3).
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Plasmid-encoded ibpA or ibpB partially suppressed a chromosomal groEL mutation leading to phage-growth deficiency. Because LS chaperonins are involved in protein-folding, prevention of protein aggregation during stress, and correct refolding after heat shock (11), this suppression is evidence that either IbpA or IbpB can replace, yet with low efficiency, some of the cellular functions carried on by the LS chaperonin.
In eukaryotes, Hsp27, Hsp25, and B-crystallines are found in protein
granules of neurodegenerative diseases and after heat shock (33-35).
In Drosophila, heat-induced granules dissolve after stress
(36). Similarly, IbpA/B proteins were found in E. coli associated with inclusion bodies and with heat-induced intracellular proteins aggregates. IbpA/B were found to be involved in the
re-solubilization of protein aggregates after stress (21). We
demonstrated here in vitro that IbpB-bound proteins are
stabilized in a conformation that can be subsequently released and
specifically refolded by the KJE chaperones and ATP. It is tempting to
speculate that stress-denatured proteins that accumulate together with
IbpA/B proteins in the E. coli cell, can be solubilized and
refolded, or degraded after stress, in processes involving the KJE
chaperones, ATP hydrolysis, and possibly other chaperones. Moreover, in
eukaryotes where heat-shock granules and amyloid plaques often contain
sHSPs (37, 35), a similar solubilization mechanism of sHSP-bound
protein aggregates, involving Hsp70 and other ATP-hydrolyzing
chaperones such as Hsp104, is therefore also conceivable as a defense
mechanism against neurodegenerative and prion diseases (38).
There are major differences in the expression level of the various families of Hsps between organisms. While heat shock induces a number of distinct Hsp families in E. coli and yeast (39), Hsp70 is by far the major heat-induced protein in Drosophila (38), and small heat-shock proteins Hsp18.1 and Hsp17.7 are the major heat-induced proteins in higher plants (3, 5). We find that small heat-shock proteins, while possibly binding non-native proteins with relatively low affinity, may yet serve as a primary protein-binding matrix for subsequent refolding by a highly cooperative multichaperone machinery. Despite their high binding efficiency, LS chaperonins may not be present in sufficient amounts to bind all non-native protein during cellular stress (39). Hence, auxiliary chaperone systems such as sHsps may become essential in vivo for the stress response.
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ACKNOWLEDGEMENTS |
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We thank C. Hergersberg for plasmids pBF3-IbpA and pBF3-IbpB, H.-J Schönfeld for purified DnaK, DnaJ, and GrpE; B. Bukau for DnaK and DnaJ producing plasmids, and G. Lorimer for discussions.
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FOOTNOTES |
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* This work was supported by a grant from the German-Israeli Foundation for Scientific Research & Development (to P. G. and J. 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{at}vms.huji.ac.il.
1 The abbreviations used are: sHsp, small heat-shock protein(s); IbpB, inclusion body associated protein B; MDH, malate dehydrogenase; LDH, lactate dehydrogenase; S, GroES; L, GroEL; K, DnaK; J, DnaJ; E, GrpE.
2 A. Azem, J. Buchner, and P. Goloubinoff, unpublished data.
3 C. Weiss, S. Gross, and P. Goloubinoff, personal communication.
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REFERENCES |
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