Krzysztof Liberek

Escherichia coli DnaJ and GrpE heat shock proteins jointly stimulate ATPase activity of DnaK.

The products of the Escherichia coli dnaK, dnaJ, and grpE heat shock genes have been previously shown to be essential for bacteriophage lambda DNA replication at all temperatures and for bacterial survival under certain conditions. DnaK, the bacterial heat shock protein hsp70 analogue and putative chaperonin, possesses a weak ATPase activity. Previous work has shown that ATP hydrolysis allows the release of various polypeptides complexed with DnaK. Here we demonstrate that the ATPase activity of DnaK can be greatly stimulated, up to 50-fold, in the simultaneous presence of the DnaJ and GrpE heat shock proteins. The presence of either DnaJ or GrpE alone results in a slight stimulation of the ATPase activity of DnaK. The action of the DnaJ and GrpE proteins may be sequential, since the presence of DnaJ alone leads to an acceleration in the rate of hydrolysis of the DnaK-bound ATP. The presence of GrpE alone increases the rate of release of bound ATP or ADP without affecting the rate of hydrolysis. The stimulation of the ATPase activity of DnaK may contribute to its more efficient recycling, and it helps explain why mutations in dnaK, dnaJ, or grpE genes often exhibit similar pleiotropic phenotypes.

Chaperones in control of protein disaggregation

The chaperone protein network controls both initial protein folding and subsequent maintenance of proteins in the cell. Although the native structure of a protein is principally encoded in its amino‐acid sequence, the process of folding in vivo very often requires the assistance of molecular chaperones. Chaperones also play a role in a post‐translational quality control system and thus are required to maintain the proper conformation of proteins under changing environmental conditions. Many factors leading to unfolding and misfolding of proteins eventually result in protein aggregation. Stress imposed by high temperature was one of the first aggregation‐inducing factors studied and remains one of the main models in this field. With massive protein aggregation occurring in response to heat exposure, the cell needs chaperones to control and counteract the aggregation process. Elimination of aggregates can be achieved by solubilization of aggregates and either refolding of the liberated polypeptides or their proteolysis. Here, we focus on the molecular mechanisms by which heat‐shock protein 70 (Hsp70), Hsp100 and small Hsp chaperones liberate and refold polypeptides trapped in protein aggregates.

Initiation of lambda DNA replication with purified host- and bacteriophage-encoded proteins: the role of the dnaK, dnaJ and grpE heat shock proteins.

Based on previous in vivo genetic analysis of bacteriophage lambda growth, we have developed two in vitro lambda DNA replication systems composed entirely of purified proteins. One is termed ‘grpE‐independent’ and consists of supercoiled lambda dv plasmid DNA, the lambda O and lambda P proteins, as well as the Escherichia coli dnaK, dnaJ, dnaB, dnaG, ssb, DNA gyrase and DNA polymerase III holoenzyme proteins. The second system includes the E.coli grpE protein and is termed ‘grpE‐dependent’. Both systems are specific for plasmid molecules carrying the ori lambda DNA initiation site. The major difference in the two systems is that the ‘grpE‐independent’ system requires at least a 10‐fold higher level of dnaK protein compared with the grpE‐dependent one. The lambda DNA replication process may be divided into several discernible steps, some of which are defined by the isolation of stable intermediates. The first is the formation of a stable ori lambda‐lambda O structure. The second is the assembly of a stable ori lambda‐lambda O‐lambda P‐dnaB complex. The addition of dnaJ to this complex also results in an isolatable intermediate. The dnaK, dnaJ and grpE proteins destabilize the lambda P‐dnaB interaction, thus liberating dnaBs helicase activity, resulting in unwinding of the DNA template. At this stage, a stable DNA replication intermediate can be isolated, provided that the grpE protein has acted and/or is present. Following this, the dnaG primase enzyme recognizes the single‐stranded DNA‐dnaB complex and synthesizes RNA primers. Subsequently, the RNA primers are extended into DNA by DNA polymerase III holoenzyme. The proposed model of the molecular series of events taking place at ori lambda is substantiated by the many demonstrable protein‐protein interactions among the various participants.

Mitochondrial Hsp78, a member of the Clp/Hsp100 family in Saccharomyces cerevisiae, cooperates with Hsp70 in protein refolding

The molecular chaperone protein Hsp78, a member of the Clp/Hsp100 family localized in the mitochondria of Saccharomyces cerevisiae, is required for maintenance of mitochondrial functions under heat stress. To characterize the biochemical mechanisms of Hsp78 function, Hsp78 was purified to homogeneity and its role in the reactivation of chemically and heat‐denatured substrate protein was analyzed in vitro. Hsp78 alone was not able to mediate reactivation of firefly luciferase. Rather, efficient refolding was dependent on the simultaneous presence of Hsp78 and the mitochondrial Hsp70 machinery, composed of Ssc1p/Mdj1p/Mge1p. Bacterial DnaK/DnaJ/GrpE, which cooperates with the Hsp78 homolog, ClpB in Escherichia coli, could not substitute for the mitochondrial Hsp70 system. However, efficient Hsp78‐dependent refolding of luciferase was observed if DnaK was replaced by Ssc1p in these experiments, suggesting a specific functional interaction of both chaperone proteins. These findings establish the cooperation of Hsp78 with the Hsp70 machinery in the refolding of heat‐inactivated proteins and demonstrate a conserved mode of action of ClpB homologs.

On the mechanism of FtsH‐dependent degradation of the σ32 transcriptional regulator of Escherichia coli and the role of the DnaK chaperone machine

The Escherichia coliσ32 transcriptional regulator has been shown to be degraded both in vivo and in vitro by the FtsH (HflB) protease, a member of the AAA protein family. In our attempts to study this process in detail, we found that two σ32 mutants lacking 15–20 C‐terminal amino acids had substantially increased half‐lives in vivo or in vitro, compared with wild‐type σ32. A truncated version of σ32, σ32CΔ, was purified to homogeneity and shown to be resistant to FtsH‐dependent degradation in vitro, suggesting that FtsH initiates σ32 degradation from its extreme C‐terminal region. Purified σ32CΔ interacted with the DnaK and DnaJ chaperone proteins in a fashion similar to that of wild‐type σ32. However, in contrast to wild‐type σ32, σ32CΔ was largely deficient in its in vivo and in vitro interaction with core RNA polymerase. As a consequence, the truncated σ32 protein was completely non‐functional in vivo, even when overproduced. Furthermore, it is shown that wild‐type σ32 is protected from degradation by FtsH when complexed to the RNA polymerase core, but sensitive to proteolysis when in complex with the DnaK chaperone machine. Our results are in agreement with the proposal that the capacity of the DnaK chaperone machine to autoregulate its own synthesis negatively is simply the result of its ability to sequester σ32 from RNA polymerase, thus making it accessible to degradation by the FtsH protease.

The small heat shock protein IbpA of Escherichia coli cooperates with IbpB in stabilization of thermally aggregated proteins in a disaggregation competent state.

The small heat shock proteins are ubiquitous stress proteins proposed to increase cellular tolerance to heat shock conditions. We isolated IbpA, the Escherichia coli small heat shock protein, and tested its ability to keep thermally inactivated substrate proteins in a disaggregation competent state. We found that the presence of IbpA alone during substrate thermal inactivation only weakly influences the ability of the bi-chaperone Hsp70-Hsp100 system to disaggregate aggregated substrate. Similar minor effects were observed for IbpB alone, the other E. coli small heat shock protein. However, when both IbpA and IbpB are simultaneously present during substrate inactivation they efficiently stabilize thermally aggregated proteins in a disaggregation competent state. The properties of the aggregated protein substrates are changed in the presence of IbpA and IbpB, resulting in lower hydrophobicity and the ability of aggregates to withstand sizing chromatography conditions. IbpA and IbpB form mixed complexes, and IbpA stimulates association of IbpB with substrate.

Distinct Activities of Escherichia coli Small Heat Shock Proteins IbpA and IbpB Promote Efficient Protein Disaggregation

It has been proposed that small heat shock proteins (sHsps) associate with aggregated proteins and change their physical properties in such a way that chaperone-mediated disaggregation and refolding become much more efficient. Here, we investigate the influence of two Escherichia coli sHsps, IbpA and IbpB, on the properties of aggregates formed under heat shock conditions and the susceptibility of these aggregates to chaperone-dependent reactivation. Our results show that the presence of IbpA during heat denaturation is sufficient to change the macroscopic properties of aggregates. The aggregates are substantially smaller than aggregates formed in the absence of sHsps and they are stained differently on electron micrographs. Moreover, these aggregates are indistinguishable, by electron microscopy studies and sedimentation analysis, from aggregates obtained during heat denaturation in the presence of IbpA and IbpB. However, the morphological similarity between these two types of aggregates does not correlate with similar susceptibility to Hsp100-Hsp70-dependent reactivation. The presence of IbpA alone during substrate denaturation does not increase the efficiency of the subsequent Hsp100-Hsp70-dependent reactivation. On the contrary, substantial inhibition of this process is observed. IbpB associates with aggregates at high temperature due to its interaction with IbpA and releases the IbpA-mediated inhibitory effect. Our results suggest there is an interplay between IbpA and IbpB in promoting Hsp100-Hsp70-mediated disaggregation of protein aggregates. Although each seems to play a different role in this process, they cooperate to stabilize protein aggregates in a disaggregation-competent state.

Both ambient temperature and the DnaK chaperone machine modulate the heat shock response in Escherichia coli by regulating the switch between sigma 70 and sigma 32 factors assembled with RNA polymerase.

In Escherichia coli individual sigma factors direct RNA polymerase (RNAP) to specific promoters. Upon heat shock induction there is a transient increase in the rate of transcription of approximately 20 heat shock genes, whose promoters are recognized by the RNAP‐sigma 32 rather than the RNAP‐sigma 70 holoenzyme. At least three heat shock proteins, DnaK, DnaJ and GrpE, are involved in negative modulation of the sigma 32‐dependent heat shock response. Here we show, using purified enzymes, that upon heat treatment of RNAP holoenzyme the sigma 70 factor is preferentially inactivated, whereas the resulting heat‐treated RNAP core is still able to initiate transcription once supplemented with sigma 32 (or fresh sigma 70). Heat‐aggregated sigma 70 becomes a target for the joint action of DnaK, DnaJ and GrpE proteins, which reactivate it in an ATP‐dependent reaction. The RNAP‐sigma 32 holoenzyme is relatively stable at temperatures at which the RNAP‐sigma 70 holoenzyme is inactivated. Furthermore, we show that formation of the RNAP‐sigma 32 holoenzyme is favored over that of RNAP‐sigma 70 at elevated temperatures. We propose a model of negative autoregulation of the heat shock response in which cooperative action of DnaK, DnaJ and GrpE heat shock proteins switches transcription back to constitutively expressed genes through the simultaneous reactivation of heat‐aggregated sigma 70, as well as sequestration of sigma 32 away from RNAP.

A Bichaperone (Hsp70-Hsp78) System Restores Mitochondrial DNA Synthesis following Thermal Inactivation of Mip1p Polymerase

Mitochondrial DNA synthesis is a thermosensitive process in the yeast Saccharomyces cerevisiae. We found that restoration of mtDNA synthesis following heat treatment of cells is dependent on reactivation of the mtDNA polymerase Mip1p through the action of a mitochondrial bichaperone system consisting of the Hsp70 system and the Hsp78 oligomeric protein. mtDNA synthesis was inefficiently restored after heat shock in yeast lacking either functional component of the bichaperone system. Furthermore, the activity of purified Mip1p was also thermosensitive; however, the purified components of the mitochondrial bichaperone system (Ssc1p, Mdj1p, Mge1p, and Hsp78p) were able to protect its activity under moderate heat shock conditions as well as to reactivate thermally inactivated Mip1p. Interestingly, the reactivation of endogenous Mip1p contributed more significantly to the restoration of mtDNA synthesis than did import of newly synthesized Mip1p from the cytosol. These observations suggest an important link between function of mitochondrial chaperones and the propagation of mitochondrial genomes under ever-changing environmental conditions.

Differential inhibition of transcription from σ70- and σ32-dependent promoters by rifampicin

Rifampicin is an antibiotic which binds to the β subunit of prokaryotic RNA polymerases and prevents initiation of transcription. It was found previously that production of heat shock proteins in Escherichia coli cells after a shift from 30°C to 43°C is not completely inhibited by this antibiotic. Here we demonstrate that while activity of a p L‐lacZ fusion (p L is a σ70‐dependent promoter) in E. coli cells is strongly inhibited by rifampicin, a p groE‐lacZ fusion, whose activity is dependent on the σ32 factor, retains significant residual activity even at relatively high rifampicin concentrations. Differential sensitivity to this antibiotic of RNA polymerase holoenzymes containing either the σ70 or the σ32 subunit was confirmed in vitro. Since the effects of an antibiotic that binds to the β subunit can be modulated by the presence of either the σ70 or the σ32 subunit in the holoenzyme, it is tempting to speculate that binding of various σ factors to the core of RNA polymerase results in different conformations of particular holoenzymes, including changes in the core enzyme.