title: DnaK prevents human insulin amyloid fiber formation on hydrophobic surfaces.
abstract: We have developed a multiwell-based protein aggregation assay to study the kinetics of insulin adsorption and aggregation on hydrophobic surfaces and to investigate the molecular mechanisms involved. Protein-surface interaction progresses in two phases: (1) a lag phase during which proteins adsorb and prefibrillar aggregates form on the material surface and (2) a growth phase during which amyloid fibers form and then are progressively released into solution. We studied the effect of three bacterial chaperones, DnaK, DnaJ, and ClpB, on insulin aggregation kinetics. In the presence of ATP, the simultaneous presence of DnaK, DnaJ, and ClpB allows good protection of insulin against aggregation. In the absence of ATP, DnaK alone is able to prevent insulin aggregation. Furthermore, DnaK binds to insulin adsorbed on hydrophobic surfaces. This process is slowed in the presence of ATP and can be enhanced by the cochaperone DnaJ. The peptide LVEALYL, derived from the insulin B chain, is known to promote fast aggregation in a concentration- and pH-dependent manner in solution. We show that it also shortens the lag phase for insulin aggregation on hydrophobic surfaces. As this peptide is also a known DnaK substrate, our data indicate that the peptide and the chaperone might compete for a common site during the process of insulin aggregation on hydrophobic surfaces.
authors.affiliation: Laboratoire des Matériaux et du Génie Physique, Grenoble Institute of Technology, 3 parvis Louis Néel, 38016 Grenoble Cedex 1, France.
authors.forename: Thomas
authors.initials: T
authors.lastname: Ballet
authors.forename: Franz
authors.initials: F
authors.lastname: Brukert
authors.forename: Paolo
authors.initials: P
authors.lastname: Mangiagalli
authors.forename: Christophe
authors.initials: C
authors.lastname: Bureau
authors.forename: Laurence
authors.initials: L
authors.lastname: Boulangé
authors.forename: Laurent
authors.initials: L
authors.lastname: Nault
authors.forename: Thomas
authors.initials: T
authors.lastname: Perret
authors.forename: Marianne
authors.initials: M
authors.lastname: Weidenhaupt
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000682:Amyloid / Q000737:chemistry* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007328:Insulin / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D018832:Molecular Chaperones / Q000737:chemistry* / Q000378:metabolism; D020673:Protein Renaturation; D013499:Surface Properties
chemical_list: D000682:Amyloid; D001426:Bacterial Proteins; D006360:Heat-Shock Proteins; D007328:Insulin; D018832:Molecular Chaperones; D000255:Adenosine Triphosphate