The Hsp70 chaperone machines of <i>Escherichia coli</i>: a paradigm for the repartition of chaperone functions

Genevaux, Pierre; Georgopoulos, Costa; Kelley, William L.

doi:10.1111/j.1365-2958.2007.05961.x

Cited by 229 publications

(235 citation statements)

References 150 publications

(231 reference statements)

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“…Hsp70/DnaK proteins have a weak ATPase activity and a peptide binding activity with a mechanism of coupling these two activities such that the addition of ATP induces peptide release [5,49]. The open/closed state of the Hsp70/ DnaK substrate-binding domain, which is mediated by the repositioning of a α-helical lid over the substrate-binding pocket, is governed by the nucleotide occupancy and status in the ATPase domain [5,50]. The conformational change induced by ATP is the switch that releases the bound peptides and denatured proteins in vivo [47,48].…”

Section: Secondary Structure Of Bldnakmentioning

confidence: 99%

A 70-kDa molecular chaperone, DnaK, from the industrial bacterium Bacillus licheniformis: gene cloning, purification and molecular characterization of the recombinant protein

et al. 2009

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The heat shock protein 70 (Hsp70/DnaK) gene of Bacillus licheniformis is 1,839 bp in length encoding a polypeptide of 612 amino acid residues. The deduced amino acid sequence of the gene shares high sequence identity with other Hsp70/DnaK proteins. The characteristic domains typical for Hsps/DnaKs are also well conserved in B. licheniformis DnaK (BlDnaK). BlDnaK was overexpressed in Escherichia coli using pQE expression system and the recombinant protein was purifi ed to homogeneity by nickel-chelate chromatography. The optimal temperature for ATPase activity of the purifi ed BlDnaK was 40°C in the presence of 100 mM KCl. The purifi ed BlDnaK had a V max of 32.5 nmol Pi/min and a K M of 439 μM. In vivo, the dnaK gene allowed an E. coli dnaK756-ts mutant to grow at 44°C, suggesting that BlDnaK should be functional for survival of host cells under environmental changes especially higher temperature. We also described the use of circular dichroism to characterize the conformation change induced by ATP binding. Binding of ATP was not accompanied by a net change in secondary structure, but ATP together with Mg 2+ and K + ions had a greater enhancement in the stability of BlDnaK at stress temperatures. Simultaneous addition of DnaJ, GrpE, and NR-peptide (NRLLLTG) synergistically stimulates the ATPase activity of BlDnaK by 11.7-fold.

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Section: Secondary Structure Of Bldnakmentioning

confidence: 99%

A 70-kDa molecular chaperone, DnaK, from the industrial bacterium Bacillus licheniformis: gene cloning, purification and molecular characterization of the recombinant protein

et al. 2009

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“…The remainder was PE-up and included seven extracellular PE-on proteins (ABC transporter periplasmic-binding protein ArtJ, hypothetical protein Lpg0264, flagellar hook protein FlgK, phospholipase C PlcB, protease/elastase LasB, polysaccharide deacetylase Lpg1993, Lpg2443) (70,(92)(93)(94). The further PE-up extr majorly included 8 proteins without assigned function (Lpg2220, Lpg1647, Lpg1645, Lpg0957, Lpg0301 Lpg1318, Lpg2206, Lpg2246), 5 T2SS substrates (chitinase ChiA, phospholipase A/acyltransferase PlaC, hypothetical protein Lpg0956, Lpg0189, Lpg0264), three Dot/Icm effectors (LegP, Lpg1667, Lpg2443) and Dot/Icm apparatus core complex protein DotC, as well as further (in addition to FlgK) motility-related proteins (FliC, FliD) (26,70,(95)(96)(97).…”

Section: Overview Of L Pneumophila E and Pe Soluble Whole Cellmentioning

confidence: 99%

Life Stage-specific Proteomes of Legionella pneumophila Reveal a Highly Differential Abundance of Virulence-associated Dot/Icm effectors

Auraß

Gerlach

Becher

et al. 2016

Molecular & Cellular Proteomics

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Major differences in the transcriptional program underlying the phenotypic switch between exponential and post-exponential growth of Legionella pneumophila were formerly described characterizing important alterations in infection capacity. Additionally, a third state is known where the bacteria transform in a viable but nonculturable state under stress, such as starvation. We here describe phase-related proteomic changes in exponential phase (E), postexponential phase (PE) bacteria, and unculturable microcosms (UNC) containing viable but nonculturable state cells, and identify phasespecific proteins. We present data on different bacterial subproteomes of E and PE, such as soluble whole cell proteins, outer membrane-associated proteins, and extracellular proteins. In total, 1368 different proteins were identified, 922 were quantified and 397 showed differential abundance in E/PE. The quantified subproteomes of soluble whole cell proteins, outer membrane-associated proteins, and extracellular proteins; 841, 55, and 77 proteins, respectively, were visualized in Voronoi treemaps. 95 proteins were quantified exclusively in E, such as cell division proteins MreC, FtsN, FtsA, and ZipA; 33 exclusively in PE, such as motility-related proteins of flagellum biogenesis FlgE, FlgK, and FliA; and 9 exclusively in unculturable microcosms soluble whole cell proteins, such as hypothetical, as well as transport/binding-, and metabolism-related proteins. A high frequency of differentially abundant or phase-exclusive proteins was observed among the 91 quantified effectors of the major virulence-associated protein secretion system Dot/Icm (> 60%). 24 were E-exclusive, such as LepA/B, YlfA, MavG, Lpg2271, and 13 were PE-exclusive, such as RalF, VipD, Lem10. The growth phase-related specific abundance of a subset of Dot/Icm virulence effectors was confirmed by means of Western blotting. We therefore conclude that many effectors are predominantly abundant at either E or PE which suggests their phase specific function. The distinct temporal or spatial presence of such proteins might have important implications for functional assignments in the future or for use as lifestage specific markers for pathogen analysis. Molecular

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“…In vitro, DnaK can perform this function when assisted by its two cochaperones, a J-domain-containing protein (either DnaJ, CbpA, or DjlA; Genevaux et al 2007) and the nucleotide exchange factor GrpE (Sharma et al 2009). Specificity of the chaperone system is conferred primarily by the DnaJ co-chaperone, which recognizes and binds to a misfolded polypeptide (Hinault et al 2010), followed by the binding of the polypeptide to a "low-affinity" DnaK, which becomes entrapped upon hydrolyzing ATP.…”

Section: Electronic Supplementary Materialsmentioning

confidence: 99%

Reactivation of protein aggregates by mortalin and Tid1—the human mitochondrial Hsp70 chaperone system

Iosefson

Sharon

Goloubinoff

et al. 2012

Cell Stress and Chaperones

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The mitochondrial 70-kDa heat shock protein (mtHsp70), also known in humans as mortalin, is a central component of the mitochondrial protein import motor and plays a key role in the folding of matrix-localized mitochondrial proteins. MtHsp70 is assisted by a member of the 40-kDa heat shock protein co-chaperone family named Tid1 and a nucleotide exchange factor. Whereas, yeast mtHsp70 has been extensively studied in the context of protein import in the mitochondria, and the bacterial 70-kDa heat shock protein was recently shown to act as an ATP-fuelled unfolding enzyme capable of detoxifying stably misfolded polypeptides into harmless natively refolded proteins, little is known about the molecular functions of the human mortalin in protein homeostasis. Here, we developed novel and efficient purification protocols for mortalin and the two spliced versions of Tid1, Tid1-S, and Tid1-L and showed that mortalin can mediate the in vitro ATP-dependent reactivation of stable-preformed heat-denatured model aggregates, with the assistance of Mge1 and either Tid1-L or Tid1-S co-chaperones or yeast Mdj1. Thus, in addition of being a central component of the protein import machinery, human mortalin together with Tid1, may serve as a protein disaggregating machine which, for lack of Hsp100/ClpB disaggregating co-chaperones, may carry alone the scavenging of toxic protein aggregates in stressed, diseased, or aging human mitochondria.

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The Hsp70 chaperone machines of Escherichia coli: a paradigm for the repartition of chaperone functions

Cited by 229 publications

References 150 publications

A 70-kDa molecular chaperone, DnaK, from the industrial bacterium Bacillus licheniformis: gene cloning, purification and molecular characterization of the recombinant protein

A 70-kDa molecular chaperone, DnaK, from the industrial bacterium Bacillus licheniformis: gene cloning, purification and molecular characterization of the recombinant protein

Life Stage-specific Proteomes of Legionella pneumophila Reveal a Highly Differential Abundance of Virulence-associated Dot/Icm effectors

Reactivation of protein aggregates by mortalin and Tid1—the human mitochondrial Hsp70 chaperone system

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