Hsp70 molecular chaperones are required to support p53 tumor suppressor activity under stress conditions.

p53 as an unstable protein in vitro likely requires stabilizing factors to act as a tumor suppressor in vivo. Here, we show that in human cells transfected with wild-type (WT) p53, Hsp90 and Hsp70 molecular chaperones maintain the p53 native conformation under heat-shock conditions (42 degrees C) as well as assist p53 refolding at 37 degrees C, during the recovery from heat shock. We also show that the interaction of WT p53 with WAF1 promoter in cells is sensitive to Hsp70 and Hsp90 inhibition already at 37 degrees C and further decreased on heat shock. The influence of chaperones on p53 binding to the WAF1 promoter sequence has been confirmed in vitro, using highly purified proteins. Hsp90 stabilizes the binding of p53 to the promoter sequence at 37 degrees C, whereas under heat-shock conditions the requirement for the Hsp70-Hsp40 system and its cooperation with Hsp90 increases. Hop co-chaperone additionally stimulates these reactions. Interestingly, the combined Hsp90 and Hsp70-Hsp40 allow for a limited in vitro restoration of the DNA-binding activity by the p53 oncogenic variant R249S and affect its conformation in cells. Our results indicate for the first time that, especially under stress conditions, not only Hsp90 but also Hsp70 is required for the chaperoning of WT and R249S p53.

Co-chaperones Bag-1, Hop and Hsp40 regulate Hsc70 and Hsp90 interactions with wild-type or mutant p53.

Using highly purified proteins, we have identified intermediate reactions that lead to the assembly of molecular chaperone complexes with wild-type or mutant p53R175H protein. Hsp90 possesses higher affinity for wild-type p53 than for the conformational mutant p53R175H. The presence of Hsp90 in a complex with wild-type p53 inhibits the binding of Hsp40 and Hsc70 to p53, consequently preventing the formation of wild-type p53-multiple chaperone complexes. The conformational mutant p53R175H can form a stable heterocomplex with Hsp90 only in the presence of Hsc70, Hsp40, Hop and ATP. The anti-apoptotic factor Bag-1 can dissociate Hsp90 from a pre- assembled complex wild-type p53 protein, but it cannot dissociate a pre-assembled p53R175H-Hsp40- Hsc70-Hop-Hsp90 heterocomplex. The results presented here provide possible molecular mechanisms that can help to explain the observed in vivo role of molecular chaperones in the stabilization and cellular localization of wild-type and mutant p53 protein.

Types and localisation of p53 gene mutations: a report on 332 non-small cell lung cancer patients.

Mutations of p53 suppressor gene are among the most common molecular abnormalities in human malignancies. We demonstrated earlier significant differences in mutational profiles between NSCLC patients from Poland and Spain. These differences were most probably related to ethnic and/or geographical factors. In the present study we analyzed the types and location of p53 gene mutations in a large group of 332 operated NSCLC patients from two institutions in Northern Poland. Within the last decades this region has been characterized by the highest incidence of lung cancer in Poland. We used both frozen and paraffin-embedded tumor samples and the screened region included exons from 5 to 8. A total of 96 samples (29%) were positive for p53 gene mutation. The proportion of mutations in particular exons was as follows: exon 5-33%, exon 6-22%, exon 7-16%, and exon 8-29%. Three 'hot spots' were located in codons 176,245 and 248. Evolutionary conserved domains were much more frequently affected than the regions outside domains. The majority of mutations (73%) were missense type, followed by null and silent mutations (21 and 6%, respectively). In all six silent mutations substituted was the third base in codon. There were no major differences in the types and locations of mutations between patients from the two institutions. This homogeneity, together with our earlier findings, may confirm the impact of ethnic and geographical factors on the mutational profile of p53 gene in NSCLC.

Structure-function analysis of the zinc-binding region of the Clpx molecular chaperone.

The ClpX heat shock protein of Escherichia coli is a member of the universally conserved Hsp100 family of proteins, and possesses a putative zinc finger motif of the C(4) type. The ClpX is an ATPase which functions both as a substrate specificity component of the ClpXP protease and as a molecular chaperone. Using an improved purification procedure we show that the ClpX protein is a metalloprotein complexed with Zn(II) cations. Contrary to other Hsp100 family members, ClpXZn(II) exists in an oligomeric form even in the absence of ATP. We show that the single ATP-binding site of ClpX is required for a variety of tasks, namely, the stabilization of the ClpXZn(II) oligomeric structure, binding to ClpP, and the ClpXP-dependent proteolysis of the lambdaO replication protein. Release of Zn(II) from ClpX protein affects the ability of ClpX to bind ATP. ClpX, free of Zn(II), cannot oligomerize, bind to ClpP, or participate in ClpXP-dependent proteolysis. We also show that ClpXDeltaCys, a mutant protein whose four cysteine residues at the putative zinc finger motif have been replaced by serine, behaves in similar fashion as wild type ClpX protein whose Zn(II) has been released either by denaturation and renaturation, or chemically by p-hydroxymercuriphenylsulfonic acid.

DjlA is a third DnaK co-chaperone of Escherichia coli, and DjlA-mediated induction of colanic acid capsule requires DjlA-DnaK interaction.

DjlA is a 30-kDa type III membrane protein of Escherichia coli with the majority, including an extreme C-terminal putative J-domain, oriented toward the cytoplasm. No other regions of sequence similarity aside from the J-domain exist between DjlA and the known DnaK (Hsp70) co-chaperones DnaJ (Hsp40) and CbpA. In this study, we explored whether and to what extent DjlA possesses DnaK co-chaperone activity and under what conditions a DjlA-DnaK interaction could be important to the cell. We found that the DjlA J-domain can substitute fully for the J-domain of DnaJ using various in vivo functional complementation assays. In addition, the purified cytoplasmic fragment of DjlA was shown to be capable of stimulating DnaK ATPase in a manner indistinguishable from DnaJ, and, furthermore, DjlA could act as a DnaK co-chaperone in the reactivation of chemically denatured luciferase in vitro. DjlA expression in the cell is tightly controlled, and even its mild overexpression leads to induction of mucoid capsule. Previous analysis showed that DjlA-mediated induction of the wca capsule operon required the RcsC/RcsB two-component signaling system and that wca induction by DjlA was lost when cells contained mutations in either the dnaK or grpE gene. We now show using allele-specific genetic suppression analysis that DjlA must interact with DnaK for DjlA-mediated stimulation of capsule synthesis. Collectively, these results demonstrate that DjlA is a co-chaperone for DnaK and that this chaperone-co-chaperone pair is implicated directly, or indirectly, in the regulation of colanic acid capsule.

Recognition, targeting, and hydrolysis of the lambda O replication protein by the ClpP/ClpX protease.

It has previously been established that sequences at the C termini of polypeptide substrates are critical for efficient hydrolysis by the ClpP/ClpX ATP-dependent protease. We report for the bacteriophage lambda O replication protein, however, that N-terminal sequences play the most critical role in facilitating proteolysis by ClpP/ClpX. The N-terminal portion of lambda O is degraded at a rate comparable with that of wild type O protein, whereas the C-terminal domain of O is hydrolyzed at least 10-fold more slowly. Consistent with these results, deletion of the first 18 amino acids of lambda O blocks degradation of the N-terminal domain, whereas proteolysis of the O C-terminal domain is only slightly diminished as a result of deletion of the C-terminal 15 amino acids. We demonstrate that ClpX retains its capacity to bind to the N-terminal domain following removal of the first 18 amino acids of O. However, ClpX cannot efficiently promote the ATP-dependent binding of this truncated O polypeptide to ClpP, the catalytic subunit of the ClpP/ClpX protease. Based on our results with lambda O protein, we suggest that two distinct structural elements may be required in substrate polypeptides to enable efficient hydrolysis by the ClpP/ClpX protease: (i) a ClpX-binding site, which may be located remotely from substrate termini, and (ii) a proper N- or C-terminal sequence, whose exposure on the substrate surface may be induced by the binding of ClpX.

Role of bacterial chaperones in DNA replication.

Studies on the involvement of chaperone proteins in DNA replication have been limited to a few replication systems, belonging primarily to the prokaryotic world. The insights gained from these studies have substantially contributed to our understanding of the eukaryotic DNA replication process as well. The finding that molecular chaperones can activate some initiation proteins before DNA synthesis has led to the more general suggestion that molecular chaperones can influence the DNA-binding activity of many proteins, including transcriptional factors involved in cell regulatory systems. The DnaK/DnaJ/GrpE molecular chaperone system became a paradigm of our understanding of fundamental processes, such as protein folding, translocation, selective proteolysis and autoregulation of the heat-shock response. Studies on the Clp ATPase family of molecular chaperones will help to define the nature of signals involved in chaperone-dependent proteins' refolding and the degradation of misfolded proteins.

Formation of the preprimosome protects lambda O from RNA transcription-dependent proteolysis by ClpP/ClpX.

Using the bacteriophage lambda DNA replication system, composed entirely of purified proteins, we have tested the accessibility of the short-lived lambda O protein to the ClpP/ClpX protease during the various stages of lambda DNA replication. We find that binding of lambda O protein to its orilambda DNA sequence, leading to the so-called "O-some" formation, largely inhibits its degradation. On the contrary, under conditions permissive for transcription, the lambda O protein bound to the orilambda sequence becomes largely accessible to ClpP/ClpX-mediated proteolysis. However, when the lambda O protein is part of the larger orilambda:O.P.DnaB preprimosomal complex, transcription does not significantly increase ClpP/ClpX-dependent lambda O degradation. These results show that transcription can stimulate proteolysis of a protein that is required for the initiation of DNA replication.

Purification and biochemical properties of Saccharomyces cerevisiae Mdj1p, the mitochondrial DnaJ homologue.

The DnaK/DnaJ/GrpE heat shock proteins of Escherichia coli constitute the prototype DnaK chaperone machine. Various studies have shown that these three proteins work synergistically in a diverse array of biological functions, including protein folding and disaggregation, proteolysis, and transport across biological membranes. We have overexpressed and purified the mitochondrial Saccharomyces cerevisiae DnaJ homologue, Mdj1pDelta55, which lacks the mitochondrial presequence, and studied its biochemical properties in well defined in vitro systems. We find that Mdj1pDelta55 interacts with DnaK as judged both by an enzyme-linked immunosorbent assay, as well as stimulation of DnaK's weak ATPase activity in the presence of GrpE. In addition, Mdj1pDelta55 not only interacts with denatured firefly luciferase on its own, but also enables DnaK to bind to it in an ATP-dependent mode. Using co-immunoprecipitation assays we can demonstrate the presence of a stable Mdj1pDelta55-luciferase-DnaK complex. However, in contrast to DnaJ, Mdj1pDelta55 does not appear to interact well with certain seemingly folded proteins, such as the sigma32 heat shock transcription factor or the lambdaP DNA replication protein. Finally, Mdj1pDelta55 can substitute perfectly well for DnaJ in the refolding of denatured firefly luciferase by the DnaK chaperone machine. These studies demonstrate that Mdj1pDelta55 has conserved most of DnaJ's known biological properties, thus supporting an analogous functional role in yeast mitochondria.

The Clp ATPases define a novel class of molecular chaperones.

The Clp ATPases were originally identified as a regulatory component of the bacterial ATP-dependent Clp serine proteases. Proteins homologous to the Escherichia coli Clp ATPases (ClpA, B, X or Y) have been identified in every organism examined so far. Recent data suggest that the Clp ATPases are not only specificity factors which help to 'present' various protein substrates to the ClpP or other catalytic proteases, but are also molecular chaperones which can function independently of ClpP. This review discusses the recent evidence that the Clp ATPases are indeed molecular chaperones capable of either repairing proteins damaged during stress conditions or activating the initiation proteins for Mu, lambda or P1 DNA replication. A mechanism is suggested to explain how the Clp ATPases 'decide' whether to repair or destroy their protein substrates.

Structure-function analysis of the Escherichia coli GrpE heat shock protein.

We have isolated various missense mutations in the essential grpE gene of Escherichia coli based on the inability to propagate bacteriophage lambda. To better understand the biochemical mechanisms of GrpE action in various biological processes, six mutant proteins were overexpressed and purified. All of them, GrpE103, GrpE66, GrpE2/280, GrpE17, GrpE13a and GrpE25, have single amino acid substitutions located in highly conserved regions throughout the GrpE sequence. The biochemical defects of each mutant GrpE protein were identified by examining their abilities to: (i) support in vitro lambda DNA replication; (ii) stimulate the weak ATPase activity of DnaK; (iii) dimerize and oligomerize, as judged by glutaraldehyde crosslinking and HPLC size chromatography; (iv) interact with wild-type DnaK protein using either an ELISA assay, glutaraldehyde crosslinking or HPLC size chromatography. Our results suggest that GrpE can exist in a dimeric or oligomeric form, depending on its relative concentration, and that it dimerizes/oligomerizes through its N-terminal region, most likely through a computer predicted coiled-coil region. Analysis of several mutant GrpE proteins indicates that an oligomer of GrpE is the most active form that interacts stably with DnaK and that the interaction is vital for GrpE biological function. Our results also demonstrate that both the N-terminal and C-terminal regions are important for GrpE function in lambda DNA replication and its co-chaperone activity with DnaK.

Structure-function analysis of the zinc finger region of the DnaJ molecular chaperone.

DnaJ is a molecular chaperone, which not only binds to its various protein substrates, but can also activate the DnaK cochaperone to bind to its various protein substrates as well. DnaJ is a modular protein, which contains a putative zinc finger motif of unknown function. Quantitation of the released Zn(II) ions, upon challenge with p-hydroxymercuriphenylsulfonic acid, and by atomic absorption showed that two Zn(II) ions interact with each monomer of DnaJ. Following the release of Zn(II) ions, the free cysteine residues probably form disulfide bridge(s), which contribute to overcoming the destabilizing effect of losing Zn(II). Supporting this view, infrared and circular dichroism studies show that the DnaJ secondary structure is largely unaffected by the release of Zn(II). Moreover, infrared spectra recorded at different temperatures, as well as scanning calorimetry, show that the Zn(II) ions help to stabilize DnaJ's tertiary structure. An internal 57-amino acid deletion of the cysteine-reach region did not noticeably affect the affinity of this mutant protein, DnaJDelta144-200, to bind DnaK nor its ability to stimulate DnaK's ATPase activity. However, the DnaJDelta144-200 was unable to induce DnaK to a conformation required for the stabilization of the DnaK-substrate complex. Additionally, the DnaJDelta144-200 mutant protein alone was unimpaired in its ability to interact with its final sigma32 transcription factor substrate, but exhibited reduced affinity toward its P1 RepA and lambdaP substrates. Finally, these in vitro results correlate well with the in vivo observed partial inhibition of bacteriophage lambda growth in a DnaJDelta144-200 mutant background.

Real time kinetics of the DnaK/DnaJ/GrpE molecular chaperone machine action.

Applying stopped-flow fluorescence spectroscopy for measuring conformational changes of the DnaK molecular chaperone (bacterial Hsp70 homologue) and its binding to target peptide, we found that after ATP hydrolysis, DnaK is converted to the DnaK*(ADP) conformation, which possesses limited affinity for peptide substrates and the GrpE cochaperone but efficiently binds the DnaJ chaperone. In the presence of DnaJ (bacterial Hsp40 homologue), the DnaK*(ADP) form is converted back to the DnaK conformation, and the resulting DnaJ-DnaK(ADP) complex binds to peptide substrates more tightly. Formation of the DnaJ(substrate-DnaK(ADP)) complex is a rate-limiting reaction. The presence of GrpE and ATP hydrolysis promotes the fast release of the peptide substrate from the chaperone complex and converts DnaK to the DnaK*(ADP) conformation. We conclude that in the presence of DnaJ and GrpE, the binding-release cycle of DnaK is stoichiometrically coupled to the adenosine triphosphatase activity of DnaK.

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.

Divergent effects of ATP on the binding of the DnaK and DnaJ chaperones to each other, or to their various native and denatured protein substrates.

Using the native proteins lambda P, lambda O, delta 32, and RepA, as well as permanently unfolded alpha-carboxymethylated lactalbumin, we show that DnaK and DnaJ molecular chaperones possess differential affinity toward these protein substrates. In this paper we present evidence that the DnaK protein binds not only to short hydrophobic peptides, which are in an extended conformation, but also efficiently recognizes large native proteins (RepA, lambda P). The best substrate for either the DnaK or DnaJ chaperone is the native P1 coded replication RepA protein. The native delta 32 transcription factor binds more efficiently to DnaJ than to DnaK, whereas unfolded alpha-carboxymethylated lactalbumin or native lambda P binds more efficiently to DnaK than to the DnaJ molecular chaperone. The presence of nucleotides does not change the DnaJ affinity to any of the tested protein substrates. In the case of DnaK, the presence of ATP inhibits, while a nonhydrolyzable ATP analogues markedly stimulates the binding of DnaK to all of these various protein substrates. ADP has no effect on these reactions. In contrast to substrate protein binding, DnaK binds to the DnaJ chaperone protein in a radically different manner, namely ATP stimulates whereas a nonhydrolyzable ATP analogue inhibits the DnaK-DnaJ complex formation. Moreover, the DnaKc94 mutant protein lacking 94 amino acids from its C-terminal domain, which still possesses at ATPase activity and forms a transient complex with protein substrates, does not interact with DnaJ protein. We conclude that the DnaK-ADP form, derived from ATP hydrolysis, possesses low affinity to the protein substrates but can efficiently interact with DnaJ molecular chaperone.

ATP hydrolysis is required for the DnaJ-dependent activation of DnaK chaperone for binding to both native and denatured protein substrates.

Using two independent experimental approaches to monitor protein-protein interactions (enzyme-linked immunosorbent assay and size exclusion high performance liquid chromatography) we describe a general mechanism by which DnaJ modulates the binding of the DnaK chaperone to various native protein substrates, e.g. lambda P, lambda O, delta 32, P1, RepA, as well as permanently denatured alpha-carboxymethylated lactalbumin. The presence of DnaJ promotes the DnaK for efficient DnaK-substrate complex formation. ATP hydrolysis is absolutely required for such DnaJ-dependent activation of DnaK for binding to both native and denatured protein substrates. Although ADP can stabilize such as an activated DnaK-protein complex, it cannot substitute for ATP in the activation reaction. In the presence of DnaJ and ATP, DnaK possesses the affinity to different substrates which correlates well with the affinity of DnaJ alone for these protein substrates. Only when the affinity of the DnaJ chaperone for its protein substrate is relatively high (e.g. delta 32, RepA) can a tertiary complex DnaK-substrate-DnaJ be detected. In the case that DnaJ binds weakly to its substrate (lambda P, alpha-carboxymethylated lactalbumin), DnaJ is only transiently associated with the DnaK-substrate complex, but the DnaK activation reaction still occurs, albeit less efficiently.

Calf thymus Hsc70 protein protects and reactivates prokaryotic and eukaryotic enzymes.

The heat-shock 70 protein (Hsp70) chaperone family is very conserved and its prokaryotic homologue, the DnaK protein, is assumed to form one of the cellular systems for the prevention and restoration of heat-induced protein denaturation. By using anti-DnaK antibodies we purified the DnaK homologue heat-shock cognate protein (Hsc70) from calf thymus to apparent homogeneity. This protein was classified as an eukaryotic Hsc70, since (i) monoclonal antibodies against eukaryotic Hsc70 recognized it, (ii) its amino-terminal sequence showed strong homology to Hsp70s from eukaryotes and, (iii) it had an intrinsic weak ATPase activity that was stimulated by various peptide substrates. We show that this calf thymus Hsc70 protein protected calf thymus DNA polymerases alpha and epsilon as well as Escherichia coli DNA polymerase III and RNA polymerase from heat inactivation and could reactivate these heat-inactivated enzymes in an ATP-hydrolysis dependent manner, likely leading to the dissociation of aggregates formed during heat inactivation. In contrast to this, DnaK protein was exclusively able to protect and to reactivate the enzymes from E.coli but not from eukaryotic cells. Finally, the addition of calf thymus DnaJ co-chaperone homologue reduced the amount of Hsc70 required for reactivation at least 10-fold.

The ClpX heat-shock protein of Escherichia coli, the ATP-dependent substrate specificity component of the ClpP-ClpX protease, is a novel molecular chaperone.

All major classes of protein chaperones, including DnaK (the Hsp70 eukaryotic equivalent) and GroEL (the Hsp60 eukaryotic equivalent) have been found in Escherichia coli. Molecular chaperones enhance the yields of correctly folded polypeptides by preventing aggregation and even by disaggregating certain protein aggregates. Previously, we identified the ClpX heat-shock protein of E. coli because it enables the ClpP catalytic protease to degrade the bacteriophage lambda O replication protein. Here we report that ClpX alone possesses all the properties expected of a molecular chaperone protein. Specifically, it can protect the lambda O protein from heat-induced aggregation, disaggregate preformed lambda O aggregates, and even promote efficient binding of lambda O to its DNA recognition sequence. A lambda O-ClpX specific protein-protein interaction can be detected either by a modified ELISA assay or through the stimulation of ClpX's weak ATPase activity by lambda O. Unlike the behaviour of the major DnaK and GroEL chaperones, ClpX requires the presence of ATP or its non-hydrolysable analogue ATP-gamma-S for efficient interaction with other proteins including the protection of lambda O from aggregation. However, ClpX's ability to disaggregate lambda O aggregates requires hydrolysable ATP. We propose that the ClpX protein is a bona fide chaperone, whose biological role includes the maintenance of certain polypeptides in a form competent for proteolysis by the ClpP protease. Furthermore, our results suggest that the ClpX protein also performs typical chaperone protein functions independent of ClpP.