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.

Peptides that form β‐sheets on hydrophobic surfaces accelerate surface‐induced insulin amyloidal aggregation

“…As shown before [6], the LVEALYL peptide accelerates HI aggregation kinetics at pH 7.3 and 37°C, only in the presence of hydrophobic surfaces (polystyrene) but not in the presence of PEG-coated surfaces preventing HI binding. The maximum effect is observed at a peptide concentration of 8.6 lM, and the lag time is reduced by 66% on average ( Table 1).…”

“…In this study, we show that the LVEALYL peptide, adsorbed on hydrophobic surfaces at sub‐stoichiometric concentrations, favors HI binding and the formation of HI amyloidal fibers. We previously showed that HI adsorbed on hydrophobic surfaces exposes a motive containing the LVEALYL peptide, which is recognized by the DnaK/DnaJ bacterial chaperones [6]. Combining these two observations indicates how HI amyloidal aggregates form on hydrophobic surfaces.…”

Degradation of Insulin Amyloid by Antibiotic Minocycline and Formation of Toxic Intermediates

“…The binding sites of antibiotics on insulin amyloids remain unclear. The stacked β-sheet structure of insulin amyloids contains hydrophobic residues 33 , and minocycline could bind to hydrophobic sites 34 . Thus, a possible hypothesis is that the binding site of minocycline is the stacked β-sheet structure.…”

Comment on “Tuning the bioactivity of bone morphogenetic protein-2 with surface immobilization strategies” by Chen et al.

“…35 The latter is the case, for instance, of hydrophobic surface-induced amyloid fibrillation of insulin. 36,37 In this case, adsorbed proteins form clusters (nuclei) on the surface which, at a critical size, induce the growth of surface-bound amyloid fibers. Upon agitation, such protein aggregates can be released in solution which can trigger further surface aggregation and sometimes aggregate growth in solution.…”