Potassium Ions and the Molecular‐Chaperone Activity of DnaK

Feifel, Bastian; Sandmeier, Erika; Schönfeld, Hans‐Joachim; Christen, Philipp

doi:10.1111/j.1432-1033.1996.0318n.x

Cited by 46 publications

(49 citation statements)

References 18 publications

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“…(i) Materials. DnaK of E. coli was expressed and purified as described previously (43,54), and its concentration was determined photometrically with a molar absorption coefficient ([cepsilon] 280 ) of 14,500 M Ϫ1 cm Ϫ1 . Purified DnaK contained Ͻ0.1 mol of nucleotide/mol DnaK and was stored in 25 mM HEPES-NaOH, 100 mM KCl, 10 mM MgCl 2 , pH 7.4 at Ϫ80°C.…”

Section: Methodsmentioning

confidence: 99%

Cadmium Causes Misfolding and Aggregation of Cytosolic Proteins in Yeast

Jacobson

Priya

Sharma

et al. 2017

Molecular and Cellular Biology

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Cadmium is a highly poisonous metal and is classified as a human carcinogen. While its toxicity is undisputed, the underlying in vivo molecular mechanisms are not fully understood. Here, we demonstrate that cadmium induces aggregation of cytosolic proteins in living Saccharomyces cerevisiae cells. Cadmium primarily targets proteins in the process of synthesis or folding, probably by interacting with exposed thiol groups in not-yet-folded proteins. On the basis of in vitro and in vivo data, we show that cadmium-aggregated proteins form seeds that increase the misfolding of other proteins. Cells that cannot efficiently protect the proteome from cadmium-induced aggregation or clear the cytosol of protein aggregates are sensitized to cadmium. Thus, protein aggregation may contribute to cadmium toxicity. This is the first report on how cadmium causes misfolding and aggregation of cytosolic proteins in vivo. The proposed mechanism might explain not only the molecular basis of the toxic effects of cadmium but also the suggested role of this poisonous metal in the pathogenesis of certain protein-folding disorders.

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Section: Methodsmentioning

confidence: 99%

Cadmium Causes Misfolding and Aggregation of Cytosolic Proteins in Yeast

Jacobson

Priya

Sharma

et al. 2017

Molecular and Cellular Biology

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“…Escherichia coli DnaK was expressed and purified as described previously (Feifel et al, 1996;Sharma et al, 2008), whilst DnaJ and GrpE (Schönfeld et al, 1995a;Schönfeld et al, 1995b) were kindly provided by Dr H. J. Schönfeld (F. HoffmannLa Roche, Basel, Switzerland). Photinus pyralis luciferase was obtained from Sigma-Aldrich.…”

Section: In Vitro Folding Assaysmentioning

confidence: 99%

Arsenite interferes with protein folding and triggers formation of protein aggregates in yeast

Jacobson

Navarrete

Sharma

et al. 2012

Journal of Cell Science

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SummarySeveral metals and metalloids profoundly affect biological systems, but their impact on the proteome and mechanisms of toxicity are not fully understood. Here, we demonstrate that arsenite causes protein aggregation in Saccharomyces cerevisiae. Various molecular chaperones were found to be associated with arsenite-induced aggregates indicating that this metalloid promotes protein misfolding. Using in vivo and in vitro assays, we show that proteins in the process of synthesis/folding are particularly sensitive to arsenite-induced aggregation, that arsenite interferes with protein folding by acting on unfolded polypeptides, and that arsenite directly inhibits chaperone activity. Thus, folding inhibition contributes to arsenite toxicity in two ways: by aggregate formation and by chaperone inhibition. Importantly, arsenite-induced protein aggregates can act as seeds committing other, labile proteins to misfold and aggregate. Our findings describe a novel mechanism of toxicity that may explain the suggested role of this metalloid in the etiology and pathogenesis of protein folding disorders associated with arsenic poisoning.

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“…Throughout this report concentrations of GrpE refer to the monomer. DnaK was purified as described (7,11,21). Its concentration was determined photometrically with a molar absorption coefficient of ⑀ 280 ϭ 14,500 M Ϫ1 cm Ϫ1 (22).…”

Section: Downloaded Frommentioning

confidence: 99%

Thermosensor Action of GrpE

Grimshaw

Jelesarov

Siegenthaler

et al. 2003

Journal of Biological Chemistry

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Temperature directly controls functional properties of the DnaK/DnaJ/GrpE chaperone system. The rate of the high to low affinity conversion of DnaK shows a non-Arrhenius temperature dependence and above ϳ40°C even decreases. In the same temperature range, the ADP/ATP exchange factor GrpE undergoes an extensive, fully reversible thermal transition (Grimshaw, J. P. A., Jelesarov, I., Schö nfeld, H. J., and Christen, P. (2001) J. Biol. Chem. 276, 6098 -6104). To show that this transition underlies the thermal regulation of the chaperone system, we introduced an intersubunit disulfide bond into the paired long helices of the GrpE dimer. The transition was absent in disulfide-linked GrpE R40C but was restored by reduction. With disulfide-stabilized GrpE, the rate of ADP/ATP exchange and conversion of DnaK from its ADP-liganded high affinity R state to the ATP-liganded low affinity T state continuously increased with increasing temperature. With reduced GrpE R40C, the conversion became slower at temperatures >40°C, as observed with wild-type GrpE. Thus, the long helix pair in the GrpE dimer acts as a thermosensor that, by decreasing its ADP/ATP exchange activity, induces a shift of the DnaK⅐substrate complexes toward the high affinity R state and in this way adapts the DnaK/DnaJ/GrpE system to heat shock conditions. Cells respond to an increase in temperature by increased synthesis of heat shock proteins (Hsps).1 Molecular chaperone systems of the Hsp70 family prevent the formation of protein aggregates and facilitate the folding of nascent polypeptide chains and denatured proteins (for comprehensive reviews, see Refs. 1 and 2). DnaK, an Hsp70 homolog of Escherichia coli, binds peptides and segments of denatured proteins in extended conformation (3, 4) and cooperates with two cohort heat shock proteins: DnaJ, an Hsp40 homolog, and GrpE (5). The DnaK/ DnaJ/GrpE chaperone system has been extensively studied in vitro at ambient temperatures (6 -12). DnaK alternates between two states, the ATP-liganded low affinity T state with fast binding and release of the substrate and the ADP-liganded high affinity R state with slow kinetics. A substrate is first bound by T state DnaK, which is then converted to the high affinity R state through DnaJ-triggered hydrolysis of DnaKbound ATP. With the assistance of GrpE, which serves as an ADP/ATP exchange factor, DnaK is reconverted from the R state into the low affinity T state, releasing the substrate.Heat shock proteins, by definition, are induced by a heat shock, i.e. a transient increase in temperature enhances the expression level of chaperones and co-chaperones. The transcription of the genes of DnaK and its co-chaperones DnaJ and GrpE is controlled by the initiation factor 32 of RNA polymerase (for a recent review, see Ref. 13). Recently, we have investigated the direct effect of elevated temperatures on the isolated DnaK/DnaJ/GrpE chaperone system. GrpE, which is an elongated homodimer both in solution (14, 15) and in crystalline form (Fig. 1 and Ref. 16), has been found to und...

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Potassium Ions and the Molecular‐Chaperone Activity of DnaK

Cited by 46 publications

References 18 publications

Cadmium Causes Misfolding and Aggregation of Cytosolic Proteins in Yeast

Cadmium Causes Misfolding and Aggregation of Cytosolic Proteins in Yeast

Arsenite interferes with protein folding and triggers formation of protein aggregates in yeast

Thermosensor Action of GrpE

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