Molecular cloning, purification and immunological responses of recombinants GroEL and DnaK from Streptococcus pyogenes

To better understand the roles of heat shock proteins in streptococcal diseases, the groEL and dnaK genes from Streptococcus pyogenes were cloned and their products (GroEL and DnaK) and derivatives (F2GroEL, F3GroEL and C1DnaK) purified as His-tagged fusion proteins. Western blot analysis of the purified proteins with sera from individuals with streptococcal diseases demonstrated that 29 out of 36 sera tested were reactive with GroEL and eight recognized DnaK. Rabbit antiserum against myosin recognized both GroEL and DnaK. Antibodies raised against purified F2GroEL and DnaK reacted with myosin in the ELISA but not in a Western immunoblot. These data indicate that the S. pyogenes GroEL and DnaK may be important immunogens during streptococcal infections. Furthermore, we provide evidence of an immunogenic relatedness of the GroEL and DnaK proteins with myosin that could play a role in the pathogenesis of streptococcal non-suppurative sequelae.     

Direct matrix-assisted laser desorption/ionization time-of-flight mass spectrometric identification of proteins on membrane detected by Western blotting and lectin blotting

We have developed new procedures to identify proteins after they are detected by Western blotting or other interactions such as lectin blotting on membranes. Our method is based on the combination of on-membrane MALDI-TOF mass spectrometry with piezoelectric chemical inkjet technology. Using this method the GroEL, FtsZ, DnaK, and GroES proteins were successfully identified from Escherichia coli after separation on two-dimensional gels, immunostaining, and on-membrane digestion. A glycoprotein detected by lectin blotting with concanavalin A was also identified using this technique.     

Limited stress response in Streptococcus pneumoniae.

In Streptococcus pneumoniae, heat shock induces the synthesis of 65-, 73-, and 84-kDa proteins, and ethanol shock induces a 104-kDa protein. In this study, the 65-, 84-, and 104-kDa proteins were identified as members of the GroEL, ClpL and alcohol dehydrogenase families, respectively, and the general properties of the stress response of S. pneumoniae to several other stresses were characterized. However, several stresses which are known to induce stress responses in Escherichia coli and Bacillus subtilis failed to induce any high molecular weight heat-shock proteins (HSPs) such as GroEL and DnaK homologues. A minor temperature shift from 30 to 37 C triggered induction of the homologues of DnaK and GroEL of E. coli. These features may provide a foundation for evaluating the role of heat-shock proteins relative to the physiology and pathogenesis of pneumococcus.     

Localization of DnaK and GroEL in Vibrio cholerae

Though the GroEL and DnaK heat shock proteins are well characterized in prokaryotes, only scanty and controversial information exist about their cellular localization. In the present study, the localization of the heat shock proteins DnaK and GroEL in normal and heat shocked cells of Vibrio cholerae, was investigated both by immunogold labeling of ultrathin sections and biochemical methods. Much of the DnaK was found to be localized at the inner membrane in unstressed cells, most probably at the Bayer's adhesion sites. Data suggested that upon heat shock, the DnaK associated with the membrane continued to remain there, but the newly synthesized DnaK appeared mostly in the cytoplasm. GroEL in both stressed and unstressed cells was found mainly in the cytoplasm.     

Formation in vitro of complexes between an abnormal fusion protein and the heat shock proteins from Escherichia coli and yeast mitochondria.

Heat shock proteins (HSPs) of the Hsp70 and GroEL families associate with a variety of cell proteins in vivo. However, the formation of such complexes has not been systematically studied. A 31-kDa fusion protein (CRAG), which contains 12 residues of cro repressor, truncated protein A, and 14 residues of beta-galactosidase, when expressed in Escherichia coli, was found in complexes with DnaK, GrpE, protease La, and GroEL. When an E. coli extract not containing CRAG was applied to an affinity column containing CRAG, DnaK, GroEL, and GrpE were selectively bound. These HSPs did not bind to a normal protein A column. DnaK, GrpE, and the fraction of GroEL could be eluted from the CRAG column with ATP but not with a nonhydrolyzable ATP analog. The ATP-dependent release of DnaK and GroEL also required Mg2+, but GrpE dissociated with ATP alone. The binding and release of DnaK and GroEL were independent events, but the binding of GrpE required DnaK. Inactivation of DnaJ, GrpE, and GroES did not affect the association or dissociation of DnaK or GroEL from CRAG. The DnaK and GrpE proteins could be eluted with 10(-6) M ATP, but 10(-4) M was required for GroEL release. This approach allows a one-step purification of these proteins from E. coli and also the isolation of the DnaK and GroEL homologs from yeast mitochondria. Competition experiments with oligopeptide fragments of CRAG showed that DnaK and GroEL interact with different sites on CRAG and that the cro-derived domain of CRAG contains the DnaK-binding site.     

Bacteriophage Ø105clz induces the GroEL-homologue protein inBacillus subtilis

Using two-dimensional polyacrylamide gel electrophoresis, the GroEL homologue ofBacillus subtilis was shown to be induced upon infection with Ø105clz, a clear plaque mutant of the temperate bacteriophage Ø105. Western blotting of one dimensional polyacrylamide gels also showed the induction of the GroEL homologue when cells were infected with Ø105clz.     

DnaK functions as a central hub in the E. coli chaperone network

Cellular chaperone networks prevent potentially toxic protein aggregation and ensure proteome integrity. Here, we used Escherichia coli as a model to understand the organization of these networks, focusing on the cooperation of the DnaK system with the upstream chaperone Trigger factor (TF) and the downstream GroEL. Quantitative proteomics revealed that DnaK interacts with at least ~700 mostly cytosolic proteins, including ~180 relatively aggregation-prone proteins that utilize DnaK extensively during and after initial folding. Upon deletion of TF, DnaK interacts increasingly with ribosomal and other small, basic proteins, while its association with large multidomain proteins is reduced. DnaK also functions prominently in stabilizing proteins for subsequent folding by GroEL. These proteins accumulate on DnaK upon GroEL depletion and are then degraded, thus defining DnaK as a central organizer of the chaperone network. Combined loss of DnaK and TF causes proteostasis collapse with disruption of GroEL function, defective ribosomal biogenesis, and extensive aggregation of large proteins.     

The impact of dnaKJ overexpression on recombinant protein solubility results from antagonistic effects on the control of protein quality

We have produced increasing levels of DnaK and its co-chaperone DnaJ along with the model VP1LAC misfolding-prone protein, to explore the role of DnaK on the management of Escherichia coli inclusion bodies. While relative solubility of VP1LAC is progressively enhanced, the heat-shock response is down-regulated as revealed by decreasing levels of GroEL. This is accompanied by an increasing yield of VP1LAC and a non-regular evolution of its insoluble fraction, at moderate levels of DnaK resulting in more abundant inclusion bodies. Also, the impact of chaperone co-expression is much more pronounced in wild type cells than in a DnaK- mutant, probably due to the different background of heat shock proteins in these cells. The involvement of DnaK in the supervision of misfolding proteins is then pictured as a dynamic balance between its immediate holding and folding activities, and the side-effect downregulation of the heat shock response though the limitation of other chaperone and proteases activities.     

DnaK/DnaJ chaperone system reactivates endogenous E. coli thermostable FBP aldolase in vivo and in vitro; the effect is enhanced by GroE heat shock proteins

Thermally aggregated, endogenous proteins in Escherichia coli cells form the S fraction, which is separable by sucrose density gradient centrifugation. To date, relatively little is known about the mechanisms of elimination of the heat-aggregated proteins from E. coli cells and the composition of the S fraction. We have identified several proteins of the S fraction using 2D-gel electrophoresis and microsequencing. A thermostable II class fructose-1,6-bisphosphate aldolase (Fda protein) appeared to be one of numerous proteins of the S fraction. Fda was purified from E. coli overproducer strain and used as a model substrate for investigation of the role of Hsps in prevention and repair of thermal denaturation of proteins both in vivo and in vitro. We found that the heat inactivation of Fda was reversible and that its reactivation in vivo and in vitro required mainly the assistance of the DnaK/DnaJ chaperone system. The dnaK756 and dnaJ259 mutations had a negative effect on the reactivation of thermally inactivated Fda. Moreover, we showed that the reactivation process in vitro was enhanced when GroEL/GroES were added together with DnaK/DnaJ. GroEL/GroES alone were inefficient in the resolubilization or reactivation of the heat-aggregated Fda. It is supposed that the denaturation of the thermostable Fda in vivo results rather from a temporary and transient deficit of Hsps than from the direct heat effect.     

Expression of a highly toxic protein, Bax, in Escherichia coli by attachment of a leader peptide derived from the GroES cochaperone

Expression of the human apoptosis modulator protein Bax in Escherichia coli is highly toxic, resulting in cell lysis at very low concentrations (Asoh, S., et al., J. Biol. Chem. 273, 11384-11391, 1998). Attempts to express a truncated form of murine Bax in the periplasm by using an expression vector that attached the OmpA signal sequence to the protein failed to alleviate this toxicity. In contrast, attachment of a peptide based on a portion of the E. coli cochaperone GroES reduced Bax's toxicity significantly and allowed good expression. The peptide, which was attached to the N-terminus, included the amino acid sequence of the mobile loop of GroES that has been demonstrated to interact with the chaperonin, GroEL. Under normal growth conditions, expression of this construct was still toxic, but generated a small amount of detectable recombinant Bax. However, when cells were grown in the presence of 2% ethanol, which stimulated overproduction of the molecular chaperones GroEL and DnaK, toxicity was reduced and good overexpression occurred. Two-dimensional gel electrophoresis analysis showed that approximately 15-fold more GroES-loop-Bax was produced under these conditions than under standard conditions and that GroEL and DnaK were elevated approximately 3-fold.     

Proteome-wide analysis of chaperonin-dependent protein folding in Escherichia coli

The E. coli chaperonin GroEL and its cofactor GroES promote protein folding by sequestering nonnative polypeptides in a cage-like structure. Here we define the contribution of this system to protein folding across the entire E. coli proteome. Approximately 250 different proteins interact with GroEL, but most of these can utilize either GroEL or the upstream chaperones trigger factor (TF) and DnaK for folding. Obligate GroEL-dependence is limited to only approximately 85 substrates, including 13 essential proteins, and occupying more than 75% of GroEL capacity. These proteins appear to populate kinetically trapped intermediates during folding; they are stabilized by TF/DnaK against aggregation but reach native state only upon transfer to GroEL/GroES. Interestingly, substantially enriched among the GroEL substrates are proteins with (betaalpha)8 TIM-barrel domains. We suggest that the chaperonin system may have facilitated the evolution of this fold into a versatile platform for the implementation of numerous enzymatic functions.     

Characterization of interactions between Escherichia coli molecular chaperones and immobilized caseins

The molecular chaperones were affinity purified with immobilized alpha-casein (45mg protein/g beads) and beta-casein columns (30 mg protein/g beads) from two heat-induced E. coli strains, NM522 and BL21. After removing nonspecifically bound proteins with 1 M NaCl, the molecular chaperones were eluted with cold water, 1 mM Mg-ATP, or 6 M urea. The eluates from affinity columns were analyzed by SDS-PAGE and Western analysis. Western analysis identified five E. coli molecular chaperones including DnaK, DnaJ, GrpE, GroEL, and GroES in eluates. Among samples, ATP eluates showed the highest chaperone purity of 80-87% followed by cold water eluates with 62-68% purity. The beta-casein column showed a higher chaperone binding capacity than the alpha-casein column. A higher concentration of chaperones was purified from strain BL21 than strain NM522 which may have been due to the lack of lon protease in the BL21 strain.     

The induction of stress proteins in three marine Vibrio during carbon starvation

Abstract The induction of DnaK and GroEL homologous proteins by heat-shock and long-term carbon starvation was studied in Vibrio vulnificus, Vibrio sp. strain S14, and Vibrio sp. strain DW1. In each Vibrio strain one protein (60 kDa) reacted with antibodies against Escherichia coli -GroEL and two proteins, DnaK (69 kDa) and Sis1 (62-60 kDa), reacted with antibodies against E. coli -Dnak. The carbon starvation elicited induction of the stress proteins was strain-specific, suggesting that the induction of stress proteins like DnaK and GroEL in marine Vibrios might not be a uniform starvation response. It appears as of these proteins, only DnaK in Vibrio sp. strain S14 remains induced after long-term carbon starvation in the three marine bacterial strains that were tested.     

Identification and partial purification of DnaK homologue from extremely halophilic archaebacteria, Halobacterium cutirubrum

The levels of synthesis of six proteins were increased at elevated growth temperature of the extremely halophilic archaebacterium Halobacterium cutirubrum. One of these proteins, with an apparent molecular mass of 97 kDa on sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS–PAGE), bound to an ATP-agarose column in the presence of 4 M NaCl, but not in the absence of salt, indicating that this protein retained its ATP-binding activity only at high salt concentration. The NH₂-terminal sequence of this protein and the internal sequences of the tryptic peptides covering 1/3 of the total number of residues coincided with that deduced from the nucleotide sequence of the dnaK gene isolated from H. cutirubrum. The results strongly suggest that this apparent 97-kDa protein is the gene product of dnaK, although the molecular mass calculated from the nucleotide sequence is only 68,495, much smaller than the value of this protein determined by SDS–PAGE. Ferguson plot analysis indicated that this protein showed anomalous mobility on SDS–PAGE. We have purified DnaK homologue to greater than 90% homogeneity with stepwise elution from an ATP-agarose column.     

Interaction between heat shock proteins and antimicrobial peptides

Drosocin, pyrrhocoricin, and apidaecin, representing the short (18-20 amino acid residues) proline-rich antibacterial peptide family, originally isolated from insects, were shown to act on a target bacterial protein in a stereospecific manner. Native pyrrhocoricin and one of its analogues designed for this purpose protect mice from bacterial challenge and, therefore, may represent alternatives to existing antimicrobial drugs. Furthermore, this mode of action can be a basis for the design of a completely novel set of antibacterial compounds, peptidic or peptidomimetic, if the interacting bacterial biopolymers are known. Recently, apidaecin was shown to enter Escherichia coli and subsequently kill bacteria through sequential interactions with diverse target macromolecules. In this paper report, we used biotin- and fluorescein-labeled pyrrhocoricin, drosocin, and apidaecin analogues to identify biopolymers that bind to these peptides and are potentially involved in the above-mentioned multistep killing process. Through use of a biotin-labeled pyrrhocoricin analogue, we isolated two interacting proteins from E. coli. According to mass spectrometry, Western blot, and fluorescence polarization, the short, proline-rich peptides bound to DnaK, the 70-kDa bacterial heat shock protein, both in solution and on the solid-phase. GroEL, the 60-kDa chaperonin, also bound in solution. Control experiments with an unrelated labeled peptide showed that while binding to DnaK was specific for the antibacterial peptides, binding to GroEL was not specific for these insect sequences. The killing of bacteria and DnaK binding are related events, as an inactive pyrrhocoricin analogue made of all-D-amino acids failed to bind. The pharmaceutical potential of the insect antibacterial peptides is underscored by the fact that pyrrhocoricin did not bind to Hsp70, the human equivalent of DnaK. Competition assay with unlabeled pyrrhocoricin indicated differences in GroEL and DnaK binding and a probable two-site interaction with DnaK. In addition, all three antibacterial peptides strongly interacted with two bacterial lipopolysaccharide (LPS) preparations in solution, indicating that the initial step of the bacterial killing cascade proceeds through LPS-mediated cell entry.     

Low temperature or GroEL/ES overproduction permits growth of Escherichia coli cells lacking trigger factor and DnaK

Escherichia coli trigger factor (TF) and DnaK cooperate in the folding of newly synthesized proteins. The combined deletion of the TF-encoding tig gene and the dnaK gene causes protein aggregation and synthetic lethality at 30 degrees C. Here we show that the synthetic lethality of DeltatigDeltadnaK52 cells is abrogated either by growth below 30 degrees C or by overproduction of GroEL/GroES. At 23 degrees C DeltatigDeltadnaK52 cells were viable and showed only minor protein aggregation. Overproduction of GroEL/GroES, but not of other chaperones, restored growth of DeltatigDeltadnaK52 cells at 30 degrees C and suppressed protein aggregation including proteins >/=60 kDa, which normally require TF and DnaK for folding. GroEL/GroES thus influences the folding of proteins previously identified as DnaK/TF substrates.     

Asymmetric segregation of heat-shock proteins upon cell division in Caulobacter crescentus

Three Caulobacter crescentus heat-shock proteins were shown to be immunologically related to the Escherichia coli heat-shock proteins GroEL, Lon and DnaK. A fourth heat-shock protein was detected with antibody to the C. crescentus RNA polymerase. This 37,000 Mr heat-shock protein might be related to the E. coli 32,000 Mr heat-shock sigma subunit. The synthesis of the major C. crescentus RNA polymerase sigma factor was not induced by heat shock. The E. coli GroEL protein and the related protein from C. crescentus were also induced by treatment with hydrogen peroxide. Like some of the proteins in the heat-shock protein families of Drosophila and yeast, the four heat-shock proteins in C. crescentus were found to be regulated developmentally under normal conditions. All four proteins were synthesized in the predivisional cell, but the progeny showed cell type-specific bias in the level of enhanced synthesis after heat shock. The 92,000 Mr Lon homolog and the 37,000 Mr RNA polymerase subunit were preferentially synthesized in the stalked cell, whereas the synthesis of the 62,000 Mr GroEL homolog was enhanced in the progeny swarmer cell. Furthermore, the four heat-shock proteins synthesized in the predivisional cell were partitioned in a specific manner upon cell division. The stalked cell, which initiates chromosome replication immediately upon division, received the Lon homolog, the DnaK homolog and the 37,000 Mr RNA polymerase subunit. The GroEL homolog, however, was distributed equally to both the stalked cell and the swarmer cell. These results provide access to the functions of C. crescentus heat-shock proteins under both normal and stress conditions. They also allow an investigation of the regulatory signals that modulate the asymmetric distribution of proteins and their subsequent cell type-specific expression in the initial stages of a developmental program.