title: Hsp70 chaperone machine remodels protein aggregates at the initial step of Hsp70-Hsp100-dependent disaggregation.
abstract: Exposure to temperatures over a certain limit leads to massive protein aggregation in the cell. Disaggregation of such aggregates is largely dependent on the Hsp100 and Hsp70 chaperones. The exact role of the Hsp70 chaperone machine (composed of DnaK, DnaJ, and GrpE) in the Hsp100-dependent process remains unknown. In this study we focused on the Hsp70 role at the initial step of the disaggregation process. Two different aggregated model substrates, green fluorescent protein (GFP) and firefly luciferase, were incubated with the Hsp70 machine resulting in efficient fragmentation of large aggregates into smaller ones. Our data suggest that the observed fragmentation is achieved first by extraction of polypeptides from aggregates in Hsp70 chaperone machine-dependent manner and not by direct fragmentation of large aggregates. In the absence of Hsp100 (ClpB) these "extracted" polypeptides were not able to fold properly and promptly reassociated into new aggregates. The extracted GFP molecules were efficiently recognized and sequestered by a molecular trap, the mutant GroEL D87K, which binds stably to unfolded but not to native polypeptides. The binding of extracted GFP molecules to the GroEL trap prevented their reaggregation. We propose that the Hsp70 machine disentangles polypeptides from protein aggregates prior to Hsp100 action.
authors.affiliation: Department of Molecular and Cellular Biology, Faculty of Biotechnology, University of Gdansk, Kladki 24, 80-822 Gdansk, Poland.
authors.forename: Szymon
authors.initials: S
authors.lastname: Zietkiewicz
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Lewandowska
authors.forename: Pawel
authors.initials: P
authors.lastname: Stocki
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Liberek
mesh_terms: D049071:Endopeptidase Clp; D029968:Escherichia coli Proteins / Q000737:chemistry; D005990:Glycerol / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism / Q000502:physiology*; D006360:Heat-Shock Proteins / Q000737:chemistry*; D008027:Light; D008156:Luciferases / Q000378:metabolism; D008954:Models, Biological; D018832:Molecular Chaperones / Q000737:chemistry; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding; D020673:Protein Renaturation; D011506:Proteins; D015800:Protozoan Proteins / Q000737:chemistry*; D012542:Scattering, Radiation; D013997:Time Factors
chemical_list: D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D010455:Peptides; D011506:Proteins; D015800:Protozoan Proteins; D049452:Green Fluorescent Proteins; D008156:Luciferases; D049071:Endopeptidase Clp; C095049:ClpB protein, Leishmania; D005990:Glycerol