Interaction of the Molecular Chaperone DNAJB6 with Growing Amyloid-beta 42 (Aβ42) Aggregates Leads to Sub-stoichiometric Inhibition of Amyloid Formation

Cecilia Månsson,Paolo Arosio,Rasha Hussein,Harm H Kampinga,Reem M Hashem,Wilbert C Boelens,Christopher M Dobson,Tuomas P.J Knowles,Sara Linse,Cecilia Emanuelsson

doi:10.1074/jbc.m114.595124

Abstract

The human molecular chaperone protein DNAJB6 was recently found to inhibit the formation of amyloid fibrils from polyglutamine peptides associated with neurodegenerative disorders such as Huntington disease. We show in the present study that DNAJB6 also inhibits amyloid formation by an even more aggregation-prone peptide (the amyloid-beta peptide, Aβ42, implicated in Alzheimer disease) in a highly efficient manner. By monitoring fibril formation using Thioflavin T fluorescence and far-UV CD spectroscopy, we have found that the aggregation of Aβ42 is retarded by DNAJB6 in a concentration-dependent manner, extending to very low sub-stoichiometric molar ratios of chaperone to peptide. Quantitative kinetic analysis and immunochemistry studies suggest that the high inhibitory efficiency is due to the interactions of the chaperone with aggregated forms of Aβ42 rather than the monomeric form of the peptide. This interaction prevents the growth of such species to longer fibrils and inhibits the formation of new amyloid fibrils through both primary and secondary nucleation. A low dissociation rate of DNAJB6 from Aβ42 aggregates leads to its incorporation into growing fibrils and hence to its gradual depletion from solution with time. When DNAJB6 is eventually depleted, fibril proliferation takes place, but the inhibitory activity can be prolonged by introducing DNAJB6 at regular intervals during the aggregation reaction. These results reveal the highly efficacious mode of action of this molecular chaperone against protein aggregation, and demonstrate that the role of molecular chaperones can involve interactions with multiple aggregated species leading to the inhibition of both principal nucleation pathways through which aggregates are able to form.

Highlights

The origins of the inhibition of DNAJB6 against amyloid formation are unknown
DNAJB6 Inhibits Fibril Formation by the amyloid ␤ peptide (A␤)42 Peptide—We investigated the effect of DNAJB6 on fibril formation by highly purified recombinant human A␤42 peptide under quiescent conditions by monitoring the intensity of Thioflavin T (ThT) fluorescence reporting on the formation of amyloid fibrils
In the presence of even a substoichiometric concentration of the molecular chaperone DNAJB6 at a molar ratio of 1:0.1 A␤42 to DNAJB6, the aggregation process was fully suppressed for 30 h (Fig. 1A) with no observable ThT signal being detected over this length of time

Summary

Background

The origins of the inhibition of DNAJB6 against amyloid formation are unknown. Results: DNAJB6 inhibits fibril formation of the A␤42 peptide from Alzheimer disease at low sub-stoichiometric molar ratios through strong binding to aggregated species. Quantitative kinetic analysis and immunochemistry studies suggest that the high inhibitory efficiency is due to the interactions of the chaperone with aggregated forms of A␤42 rather than the monomeric form of the peptide This interaction prevents the growth of such species to longer fibrils and inhibits the formation of new amyloid fibrils through both primary and secondary nucleation. Depleted, fibril proliferation takes place, but the inhibitory activity can be prolonged by introducing DNAJB6 at regular intervals during the aggregation reaction These results reveal the highly efficacious mode of action of this molecular chaperone against protein aggregation, and demonstrate that the role of molecular chaperones can involve interactions with multiple aggregated species leading to the inhibition of both principal nucleation pathways through which aggregates are able to form. We demonstrate by means of kinetic analysis and immunochemistry experiments that such high efficiency originates from the capability of DNAJB6 to sequester effectively the A␤42 aggregates, which propagate the amyloid conversion reaction, thereby preventing their growth and limiting their ability to proliferate through secondary nucleation

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION