Disease relevance of dnaK

• Mutations in dnaK have been previously shown to affect both DNA and RNA synthesis in E. coli [1].
• The Escherichia coli dnaK gene product, originally defined by mutations that blocked lambda phage DNA replication, is known to be necessary for E. coli viability [2].
• The dnaK protein was purified by following its ability to complement the replication of single-stranded M13 bacteriophage DNA in a reaction system dependent on the presence of the lambda O and P DNA replication proteins [3].
• Post-transcriptional regulation of the Bacillus subtilis dnaK operon [4].
• Nucleotide sequence of a Bacillus megaterium gene homologous to the dnaK gene of Escherichia coli [5].

High impact information on dnaK

• Cell survival under severe thermal stress requires the activity of the ClpB (Hsp104) AAA+ chaperone that solubilizes and reactivates aggregated proteins in concert with the DnaK (Hsp70) chaperone system [6].
• A role for DnaK, the major E. coli Hsp70, in chaperoning de novo protein folding has remained elusive [7].
• Genetic evidence indicates central roles for Hsp70 chaperones in the regulation of heat shock gene expression [8].
• The dnaK protein modulates the heat-shock response of Escherichia coli [1].
• Addition of Mg-ATP results in the dissociation of the substrate polypeptides from the chaperone, but as ATP-gamma S (an ATP analogue that is only slowly hydrolysable) cannot substitute for ATP in this reaction, it has been concluded that ATP hydrolysis is necessary to dissociate Hsp70-substrate protein complexes [9].

Chemical compound and disease context of dnaK

• NH2-terminal sequence analysis revealed the 72-kDa protein to have 100% identity with the first 13 amino acid residues of the Escherichia coli dnaK heat shock protein [10].
• In vivo, elevated proline pools in Escherichia coli (obtained by altering the feedback inhibition by proline of gamma-glutamylkinase, the first enzyme of the proline biosynthesis pathway) restore the viability of a dnaK-deficient mutant at 42 degrees C, suggesting that proline can act as a thermoprotectant for E. coli cells [11].
• The product of the dnaK gene has been identified on SDS polyacrylamide gels after infection of UV-irradiated E. coli cells [12].
• When the A6-d-ANase gene was expressed in the Escherichia coli dnaK mutant dnaK756, little activity was observed in the soluble fraction, and it was restored by the coexpression of DnaKJE or the substitution of the R354 residue of A6-d-ANase for lysine [13].
• 5. The chemical analysis of the phosphorylated moiety of dnaK protein showed that it was modified exclusively at serine residues during normal growth of cells, and mostly at threonine residues after phage infection [14].

Biological context of dnaK

• In addition, bacteria that overproduce the dnaK protein at all temperatures undergo a drastically reduced heat-shock response at high temperature [1].
• Initiation of lambda DNA replication with purified host- and bacteriophage-encoded proteins: the role of the dnaK, dnaJ and grpE heat shock proteins [15].
• We have identified promoters for the Escherichia coli heat shock operons dnaK and groE and the gene encoding heat shock protein C62 [16].
• The primary DNA sequence of the entire protein-coding region of the dnaK gene was determined and compared with that of the Hsp70 gene of Drosophila [17].
• In contrast to Drosophila and Saccharomyces cerevisiae, both of which contain multigene families related to the Hsp70 gene, hybridization analyses indicate that E. coli contains only a single Hsp70-related gene, dnaK [17].

Anatomical context of dnaK

• The fusion protein, which co-purifies with the bacterial chaperones dnaK and groEL, binds to glyoxysomes and is partially translocated in an ATP-dependent reaction which is independent of eukaryotic cytosol [18].
• These abnormal ribosomal particles are rescued if the mutant cells are returned to 30 degrees C. Thus, the product of the dnaK gene is implicated in ribosome biogenesis at high temperature [19].
• These enzymes were synthesized mostly in soluble, fully enzymatically active forms in wild-type E. coli cells cultured in the temperature range 20-42 degrees C, but aggregated extensively in dnaK null mutants [20].
• Using non-pathogenic Escherichia coli strains and the human monocytic cell line U937, we showed that deletion of the dnaK gene significantly increased the rate of initial intracellular killing of bacteria [21].
• Immediately after phagocytosis, the number of viable dnaK mutant bacteria found within macrophages was significantly lower compared to that of intracellular wild type bacteria [22].

Associations of dnaK with chemical compounds

• We analyzed the effects of dnaK mutations which alter the corresponding glutamate-171 of DnaK to alanine, leucine or lysine [23].
• In addition, after incubating cells with [32P]orthophosphate at 42 degrees C, the 32P-labeled dnaK bound quantitatively to the CRAG column, unlike the nonlabeled protein [24].
• The chemical chaperone proline relieves the thermosensitivity of a dnaK deletion mutant at 42 degrees C [11].
• In the presence of stress, heat or ethanol (4%), the X. campestris pv. campestris 17 grpE and dnaK promoters were induced two- to three-fold over controls [25].
• 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 [26].

Regulatory relationships of dnaK

• Hsc66 from Escherichia coli is a constitutively expressed hsp70 class molecular chaperone whose activity is coupled to ATP binding and hydrolysis [27].

Other interactions of dnaK

• First step of this disassembling reaction is the binding of dnaK protein to lambda P protein [28].
• While dnaK and tig are the essential components for nascent polypeptide folding in E. coli, deletion did not confer synthetic lethality in B. subtilis, suggesting that under normal growth conditions, another system or mechanism with a specific role prevails [29].
• Steady-state experiments revealed that Hsc66 has a low affinity for ATP (K(m)(ATP) = 12.7 microM) compared with other hsp70 chaperones [27].
• Heat stress at 42.5 degrees C appeared to be the optimum temperature for HSP formation in cells grown at 30 degrees C. The relative rate of synthesis of HSP70 and HSP15 reached a maximum at 30 min after the temperature shift-up whereas the capability of cells to accumulate HSP64 and HSP14 continued through 2 h [30].
• Hsc66, a stress-70 protein, and Hsc20, a J-type accessory protein, comprise a newly described Hsp70-type chaperone system in addition to DnaK-DnaJ-GrpE in Escherichia coli [31].

Analytical, diagnostic and therapeutic context of dnaK

• This modification appears to be phosphorylation; after treatment with phosphatases, the ATP-eluted dnaK resembled the predominant form in electrophoretic and binding properties [24].
• The DnaK epitope was determined by Western blot analysis of a series of truncated DnaK fragments overproduced in Escherichia coli using 5' and 3' dnaK-deleted expression plasmids [32].
• A fragment of the dnaK gene was amplified from these strains by PCR with oligonucleotide primers targeting regions of the dnaK gene that are conserved at the amino acid level, and the resulting PCR products were cloned into a plasmid vector [33].
• Furthermore, analysis of aggregated proteins in the dnaK-deficient strain at 42 degrees C by two-dimensional gel electrophoresis shows that high proline pools reduce the protein aggregation defect of the dnaK-deficient strain [11].
• The results of HspR titration experiments, where the dnaK operon promoter region was cloned at ca. 50 copies per chromosome, were consistent with the prediction that HspR functions as a negative autoregulator [34].

References