Proteinaceous Infectious Behavior in Non-pathogenic Proteins Is Controlled by Molecular Chaperones

Ben‐Zvi, Anat; Goloubinoff, Pierre

doi:10.1074/jbc.m209163200

Cited by 38 publications

(30 citation statements)

References 40 publications

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“…This behavior is contrary to a classic enzymatic reaction, in which substrate dilution may only maintain or reduce the specific activity of a fixed concentration of enzyme. The observed increase in the chaperonespecific activity could not be attributed to dilution-induced changes in the substrate, as heat-denatured G6PDH aggregates are not dynamic (10,11). Rather, the specific refolding activity of the chaperone appeared to depend on its ratio with the substrate.…”

Section: Cooperation Between Dnak Molecules Is Required For Refolding Ofmentioning

confidence: 83%

“…Aggregates-When the model enzyme G6PDH is heatdenatured (52°C, 15 min) in the absence of chaperones, it forms stable inactive aggregates, mostly soluble at low-speed centrifugation (11). As initially shown by Diamant et al (10), such aggregates are very stable.…”

Section: Cooperation Between Dnak Molecules Is Required For Refolding Ofmentioning

confidence: 88%

“…The enzymatic activity of G6PDH was measured at 25°C as described previously (11). All of the protein concentrations are expressed in protomers, regardless of the oligomeric state of the chaperones or the aggregates.…”

Section: Methodsmentioning

confidence: 99%

“…The results in Fig. 2, a and b, are expressed as the fraction (%) of soluble protein after centrifugation compared with the total protein amount before centrifugation as described previously (11).…”

Section: Methodsmentioning

confidence: 99%

“…1b). Whereas in the absence of chaperones or ATP dilution had no effect on the stability and reactivity of the aggregates (10,11), the presentation of diluted aggregates to a constant high concentration of DnaK (and of DnaJ, GrpE, ClpB, and ATP) resulted in a net increase of the chaperone-specific refolding activity. At equimolar (protomer to protomer) amounts of chaperone/substrate, the specific refolding activity was very low but it became optimal when the molar ratio was restored by aggregate dilution to a 5-fold chaperone excess (Fig.…”

Section: Cooperation Between Dnak Molecules Is Required For Refolding Ofmentioning

confidence: 95%

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Active Solubilization and Refolding of Stable Protein Aggregates By Cooperative Unfolding Action of Individual Hsp70 Chaperones

Ben‐Zvi

Rios

Dietler

et al. 2004

Journal of Biological Chemistry

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113

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Hsp70 is a central molecular chaperone that passively prevents protein aggregation and uses the energy of ATP hydrolysis to solubilize, translocate, and mediate the proper refolding of proteins in the cell. Yet, the molecular mechanism by which the active Hsp70 chaperone functions are achieved remains unclear. Here, we show that the bacterial Hsp70 (DnaK) can actively unfold misfolded structures in aggregated polypeptides, leading to gradual disaggregation. We found that the specific unfolding and disaggregation activities of individual DnaK molecules were optimal for large aggregates but dramatically decreased for small aggregates. The active unfolding of the smallest aggregates, leading to proper global refolding, required the cooperative action of several DnaK molecules per misfolded polypeptide. This finding suggests that the unique ATP-fueled locking/unlocking mechanism of the Hsp70 chaperones can recruit random chaperone motions to locally unfold misfolded structures and gradually disentangle stable aggregates into refoldable proteins.

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Section: Cooperation Between Dnak Molecules Is Required For Refolding Ofmentioning

confidence: 83%

Section: Cooperation Between Dnak Molecules Is Required For Refolding Ofmentioning

confidence: 88%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Cooperation Between Dnak Molecules Is Required For Refolding Ofmentioning

confidence: 95%

See 3 more Smart Citations

Active Solubilization and Refolding of Stable Protein Aggregates By Cooperative Unfolding Action of Individual Hsp70 Chaperones

Ben‐Zvi

Rios

Dietler

et al. 2004

Journal of Biological Chemistry

Self Cite

100

113

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Acceleration of protein aggregation by amphiphilic peptides: Transformation of supramolecular structure of the aggregates

Artemova

Stein-Margolina

Bumagina

et al. 2011

Biotechnology Progress

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Protein self-assembly and aggregation represent a special tool in biomedicine and biotechnology to produce biological materials for a wide range of applications. The protein aggregates are very different morphologically, varying from soluble amorphous aggregates to highly ordered amyloid-like fibrils, the latter being associated with molecular structures able to perform specific functions in living systems. Fabrication of novel biomaterials resembling natural protein assemblies has awakened interest in identification of low-molecular-weight biogenic agents as regulators of transformation of aggregation-prone proteins into fibrillar structures. Short amphiphilic peptides can be considered for this role. Using dynamic light scattering, turbidimetry, fluorescence spectroscopy, and transmission electron microscopy (TEM), we have demonstrated that the Arg-Phe dipeptide dramatically accelerates the aggregation of a model protein, α-lactalbumin, to generate morphologically different structures. TEM revealed transformation of spherical particles observed in the control samples into branched chains of fibril-like nanostructures in the presence of the peptide, suggesting that amphiphilic peptides can induce changes in the physicochemical properties of a protein substrate (net charge, hydrophobicity, and tendency to β-structure formation) resulting in accumulation of peptide-protein complexes competent to self-assembly into supramolecular structures. A number of other short amphiphilic peptides have also been shown to accelerate the aggregation process, using alternative complementary protein substrates for identification of molecular recognition modules. Peptide-protein assemblies are suggested to play the role of building blocks for formation of supramolecular structures profoundly differing from those of the individual protein substrate in type, size, and shape.

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Comparative analysis of protein aggregates by blue native electrophoresis and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a three-dimensional geometry gel

et al. 2005

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We describe the comparative analysis of protein aggregates by combining blue native electrophoresis and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a 3-D geometry gel for simultaneous processing of many samples. The first native electrophoresis step, separating the aggregates, is carried out for a series of samples in parallel lanes within a slab gel. This gel is then placed on the top surface of a cylindrical, 3-D geometry gel for the second denaturing electrophoresis step, separating the proteins composing the aggregates. The samples migrate parallel to the vertical axis of the gel cylinder. Data are acquired online by photodetection of laser-induced fluorescence during electrophoresis. For this purpose, the samples are fluorescently labeled within the slab gel after the first separation step. A 3-D geometry gel separates the equivalent of many conventional SDS slab gels represented by vertical layers in the 3-D gel body. In this way, many samples are analyzed in the same gel under identical conditions, improving comparability and resolution and making the process considerably more efficient. This novel technique allowed the identification of several aggregate classes of recombinant proteins expressed in bacteria. We observed that proteins preferentially bind to homolog polypeptides, but also seem to form a trapping mesh co-aggregating with other proteins. The aggregation pattern revealed by this technique supplements data obtained from standard two-dimensional gel electrophoresis analysis. We expect interesting applications, for instance in aggregate monitoring of clinical samples. It should be feasible to quickly gain a diagnostic picture during amyloid-related neurodegenerative disease development or to observe drug effects on protein aggregation.

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Proteinaceous Infectious Behavior in Non-pathogenic Proteins Is Controlled by Molecular Chaperones

Cited by 38 publications

References 40 publications

Active Solubilization and Refolding of Stable Protein Aggregates By Cooperative Unfolding Action of Individual Hsp70 Chaperones

Active Solubilization and Refolding of Stable Protein Aggregates By Cooperative Unfolding Action of Individual Hsp70 Chaperones

Acceleration of protein aggregation by amphiphilic peptides: Transformation of supramolecular structure of the aggregates

Comparative analysis of protein aggregates by blue native electrophoresis and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a three-dimensional geometry gel

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