Hsc62, Hsc56, and GrpE, the third Hsp70 chaperone system of Escherichia coli

Yoshimune, Kazuaki; Yoshimura, Teizo; Nakayama, Tôru; Nishino, Tokuzo; Esaki, Nobuyoshi

doi:10.1016/s0006-291x(02)00403-5

Cited by 25 publications

(23 citation statements)

References 35 publications

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“…Thus, GrpE facilitates DnaK recycling (Bukau & Horwich, 1998). It was found recently that GrpE also stimulates the ATPase activity of Hsc62, one of the Hsp70 E. coli homologues (Yoshimune et al, 2002).…”

Section: Introductionmentioning

confidence: 96%

Aggregation of heat-shock-denatured, endogenous proteins and distribution of the IbpA/B and Fda marker-proteins in Escherichia coli WT and grpE280 cells

et al. 2004

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Submission of wild-type Escherichia coli to heat shock causes an aggregation of cellular proteins. The aggregates (S fraction) are separable from membrane fractions by ultracentrifugation in a sucrose density gradient. In contrast, no protein aggregation was detectable in an E. coli grpE280 mutant either by this technique or by electron microscopy. In search of an explanation for this observation at a molecular level, two kinds of marker proteins were used: Fda (fructose-1,6-biphosphate aldolase), the previously identified S fraction component, and IbpA/B, small heat-shock proteins abundantly associated with the S fraction proteins. Both types of marker proteins, normally never found in the outer-membrane (OM) fraction of WT cells, were present in the OM fraction from grpE cells after heat shock. This pointed to the presence of aggregates smaller than those in WT cells that cosedimented with the OM fraction. The OM fraction was enlarged in grpE cells. Although not proven directly, the presence of still smaller aggregates, not exceeding the solubility level and containing inactive Fda, was noted in the soluble CP fraction containing the cytoplasmic and periplasmic proteins. Therefore, aggregation occurred in both strains, but in a different way. The autoregulation of the heat-shock response causes a greater increase of DnaK/DnaJ and IbpAB levels in grpE cells than in WT after temperature elevation. This may explain the prevalence of the small-sized aggregates in the grpE cells. Estimation of total Fda protein before and after heat shock did not show any loss. This indicated that renaturation rather than proteolysis underlies the final disappearance of the aggregates. Though surprising at first, this is not contradictory with the participation of heat-shock proteases in removal of protein components of the S fraction as shown before, since proteins that are irreversibly denatured are probably substrates for the proteases.

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Section: Introductionmentioning

confidence: 96%

Aggregation of heat-shock-denatured, endogenous proteins and distribution of the IbpA/B and Fda marker-proteins in Escherichia coli WT and grpE280 cells

et al. 2004

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“…The rather different hemolytic activities of the compounds gave an impression of the importance of the latter effects even for bacterial cells. Alternatively, two homologues of DnaK, HscA and HscC, could be also important for mediating antibacterial activity (31,46).…”

Section: Discussionmentioning

confidence: 99%

Fatty Acyl Benzamido Antibacterials Based on Inhibition of DnaK-catalyzed Protein Folding

Liebscher

Jahreis

Lücke

et al. 2007

Journal of Biological Chemistry

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We have reported that the hsp70 chaperone DnaK from Escherichia coli might assist protein folding by catalyzing the cis/ trans isomerization of secondary amide peptide bonds in unfolded or partially folded proteins. In this study a series of fatty acylated benzamido inhibitors of the cis/trans isomerase activity of DnaK was developed and tested for antibacterial effects in E. coli MC4100 cells. N ␣ -[Tetradecanoyl-(4-aminomethylbenzoyl)]-L-asparagine is the most effective antibacterial with a minimal inhibitory concentration of 100 ؎ 20 g/ml. The compounds were shown to compete with fluorophore-labeled 32 -derived peptide for the peptide binding site of DnaK and to increase the fraction of aggregated proteins in heat-shocked bacteria. Despite its inability to serve as a folding helper in vivo a DnaK-inhibitor complex was still able to sequester an unfolded protein in vitro. Structure activity relationships revealed a distinct dependence of DnaK-assisted refolding of luciferase on the fatty acyl chain length, whereas the minimal inhibitory concentration was most sensitive to the structural nature of the benzamido core. We conclude that the isomerase activity of DnaK is a major survival factor in the heat shock response of bacteria and that small molecule inhibitors can lead to functional inactivation of DnaK and thus will display antibacterial activity.The rapid emergence of bacterial strains that are resistant to current antibiotics requires the development of antimicrobial compounds based on novel mechanisms of action. De novo protein synthesis and assisted protein folding are processes essential for viability of cells thus representing attractive targets for the design of antibacterials. The hsp70 chaperone DnaK is a folding helper enzyme showing ATPase and secondary amide peptide bond cis/trans isomerase (APIase) 2 activity that assists together with its co-chaperones the de novo protein folding of ϳ20% of the Escherichia coli proteins larger than 30 kDa (1-4). Whereas the deletion of the DnaK gene alone is not deleterious for E. coli cells, combined deletion of the ribosome-associated peptidylprolyl-cis/trans-isomerase trigger factor and DnaK genes is lethal under normal growth conditions (4, 5). Furthermore, DnaK is essential under various stress conditions like heat, oxidative stress, and nutritional deprivation (6 -8). In E. coli cells lacking DnaK, heat shock treatment at 42°C causes strong protein aggregation comprising 150 -200 different protein species and ϳ10% of the amount of pre-existing E. coli proteins (9). During infection pathogens encounter stress conditions generated by the host defense mechanisms to eliminate the infection. They respond by the rapid acceleration in the rate of expression of heat shock and other stress proteins (10). The DnaK/DnaJ chaperone machinery of Salmonella enterica has been shown to be involved in bacterial invasion of epithelial cells, and the DnaK/DnaJ-depleted mutant could not survive or proliferate at all within macrophages (11). In the case of the pathogen Brucel...

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“…Escherichia coli strain JM109 or BL21 harboring the N-acyl-D-amino acid amidohydrolase expression plasmid, alone or together with pKJE7 (DnaKJE), pTF16 (TF), pGro7 (GroELS), pGKJE8 (DnaKJE+GroELS) or pG-Tf2 (TF+GroELS) was grown at 30°C in LB medium with (+) or without ()) inducer for expression of chaperones. Data are the average of more than two determinants Inducer for chaperones N-Acetyl-D-amino acid amidohydrolase activity (U/mg) Chaperones induced by 2 mg/ml L-arabinose 5 ng/ml tetracycline c Chaperones induced by 5 ng/ml tetracycline ficities [3,4,9,33,34]. These chaperones are candidates for chaperones that might increase active N-acyl-Damino acid amidohydrolases upon their coexpression.…”

Section: Discussionmentioning

confidence: 99%

Molecular chaperones facilitate the soluble expression of N-acyl-d-amino acid amidohydrolases in Escherichia coli

Yoshimune

Ninomiya

Wakayama

et al. 2004

J IND MICROBIOL BIOTECHNOL

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The overproduction of D-aminoacylase ( D-ANase, 233.8 U/mg), N-acyl-D-glutamate amidohydrolase (D-AGase, 38.1 U/mg) or N-acyl-D-aspartate amidohydrolase (D-AAase, 6.2 U/mg) in Escherichia coli is accompanied by aggregation of the overproduced protein. To facilitate the expression of active enzymes, the molecular chaperones GroEL-GroES (GroELS), DnaK-DnaJ-GrpE (DnaKJE), trigger factor (TF), GroELS and DnaKJE or GroELS and TF were coexpressed with the enzymes. D-ANase (313.3 U/mg) and D-AGase (95.8 U/mg) were overproduced in an active form at levels 1.3- and 1.8-fold higher, respectively, upon co-expression of GroELS and TF. An E. coli strain expressing the D-AAase gene simultaneously with the TF gene exhibited a 4.3-fold enhancement in d-AAase activity (32.0 U/mg) compared with control E. coli expressing the D-AAase gene alone.

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Hsc62, Hsc56, and GrpE, the third Hsp70 chaperone system of Escherichia coli

Cited by 25 publications

References 35 publications

Aggregation of heat-shock-denatured, endogenous proteins and distribution of the IbpA/B and Fda marker-proteins in Escherichia coli WT and grpE280 cells

Aggregation of heat-shock-denatured, endogenous proteins and distribution of the IbpA/B and Fda marker-proteins in Escherichia coli WT and grpE280 cells

Fatty Acyl Benzamido Antibacterials Based on Inhibition of DnaK-catalyzed Protein Folding

Molecular chaperones facilitate the soluble expression of N-acyl-d-amino acid amidohydrolases in Escherichia coli

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