Interaction of the molecular chaperone DNAJB6 with growing amyloid-beta 42 (Aβ42) aggregates leads to sub-stoichiometric inhibition of amyloid formation

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.

Keywords: Aggregation Kinetics; Alzheimer Disease; Amyloid Fibril Formation; Amyloid-beta (Aβ); Chaperone DnaJ (DnaJ); Hsp40; Inhibition Mechanism; Neurodegenerative Disease; Protein Aggregation.

FIGURE 1.

Aβ42 aggregation in the absence and presence of the human molecular chaperone DNAJB6. A, fibril formation by 3 μm Aβ42 solutions was monitored by following the increase in ThT fluorescence (black); the inhibitory effect of DNAJB6 (red) was compared with that of human αB-crystallin (blue) and HAS (green). Molar ratios between Aβ42 and the proteins were 1:0.1. B, aggregation reaction profiles of ThT fluorescence of Aβ42 in the absence (black) and in the presence of DNAJB6 at molar ratios of peptide to chaperone from 1:0.01 to 1:0.13, color coded as indicated to the right. 4 independent incubations per sample were performed. C, silver-stained SDS-PAGE shows the purity of the chaperone with and without the washing step with 8 m urea to remove proteins strongly bound to DNAJB6: lane 1, molecular weight marker (kDa); lane 2, urea-washed DNAJB6; lane 3, not urea-washed DNAJB6; lanes 4 and 5: first and second wash fraction (with and without urea, respectively) resulting in DNAJB6 in lane 2; lane 6, wash fraction without urea resulting in DNAJB6 in lane 3. The asterisks are positioned above bands indicating minor amounts of DNAJB6 degradation product (***), DNAJB6 monomers with His-tag (**), and DNAJB6 dimers (*).

FIGURE 2.

DNAJB6 delays the conversion of Aβ42 from unstructured monomers to β-sheet structures. Far-UV CD spectra of 5 μm Aβ42 solutions showing the appearance of the characteristic β-sheet structure signal with a minimum intensity at c.a. 218 nm at different time points at 37 °C in A the absence and B, the presence of 0.05 μm DNAJB6. In A the signal from the buffer has been subtracted, in B the signal from the buffer including 0.05 μm DNAJB6 has been subtracted.

FIGURE 3.

Kinetics of Aβ42 aggregation in the presence of DNAJB6. Reaction profiles for ThT fluorescence for unseeded aggregation of (A) 3 μm Aβ42 solutions in the absence of DNAJB6 (red) and with DNAJB6 present at a molar ratio of peptide to chaperone of 1:0.001 (green), 1:0.005 (maroon) and 1:0.01 (purple), and (B) Aβ42 solutions in the concentration range 2.2–8.0 μm in the presence of 0.225 μm DNAJB6. The lines in A and B represent the integrated rate law for Aβ42 aggregation fitted to the experimental data. C, half-times as a function of the initial Aβ42 concentration in the presence and absence of DNAJB6. Note the change of exponent from −1.33 to −1.45 induced by the presence of DNAJB6, showing the capability of the chaperone to suppress primary nucleation events. D and E, set of microscopic rate constants corresponding to the kinetic analysis shown in A and B normalized to the values in the absence of chaperone as a function of DNAJB6:Aβ42 molar ratios. For unseeded reactions the inhibition of primary nucleation events (k_nk₊/k_nk*₊ (D)) is significantly larger with respect to secondary nucleation events (k₂k₊/k₂k*₊ (E)). F, reaction profiles of 3 μm Aβ42 solutions with 1% seeds in the absence of DNAJB6 (red) and with 1:0.01 (blue), 1:0.02 (green), and 1:0.05 (orange) monomer equivalents of DNAJB6 showing the capability of the chaperone to suppress secondary nucleation events.

FIGURE 4.

Prevention of nucleation pathways by the chaperone DNAJB6. Schematic diagram showing (A) the molecular pathways involved in Aβ42 aggregation and (B) the proposed mechanism by which DNAJB6 inhibits the aggregation reaction. As indicated in faded colors in B, the interactions between the chaperone and the Aβ42 growing aggregates inhibit both primary (as seen in Fig. 3A) and secondary (as seen in Fig. 3F) nucleation pathways.

FIGURE 5.

Incorporation of DNAJB6 into fibrils. Fibril formation by 3 μm Aβ42 solutions in the absence (A) and presence of 0.04 μm (B), 0.06 μm (C), and 0.07 μm (D) DNAJB6 were incubated at 37 °C and monitored by ThT fluorescence. Six samples (marked with asterisks) taken at six time points during the fibril formation process, and one sample withdrawn at the end of the experiments after 24 h, were examined. SDS-insoluble species were trapped on a cellulose acetate membrane, incubated with antibodies against Aβ42 and DNAJB6, and detected simultaneously with secondary antibodies labeled with different chromophores.

FIGURE 6.

Arrest of ongoing reactions by binding of DNAJB6 to Aβ42 aggregates. A, 0.3 μm DNAJB6 was added to 3 μm Aβ42 solutions at 0, 15, 30, 45, 60, 75, or 90 min after the initialization of fibril formation at 37 °C. B, SDS-insoluble fibrils from the reactions in A corresponding to incubation times of 45, 60, 75, and 90 min were withdrawn after 35 h in the platereader and trapped on a cellulose acetate membrane, incubated with antibodies against Aβ42 and DNAJB6, and detected simultaneously with secondary antibodies labeled with different chromophores. C, control experiments for the dual immunodetection: 1: 3 μm Aβ42 fibrils alone, 2: 3 μm Aβ42 fibrils mixed with 0.3 μm DNAJB6 just prior to trapping, and 3: 0.3 μm DNAJB6 alone.

FIGURE 7.

Extension of the inhibitory effect for longer times by multiple additions of DNAJB6. Aliquots of DNAJB6, each giving a concentration of 0.015 μm, were added repeatedly into the (A) unseeded and (B) seeded aggregation reactions, from one to seven times at the time points 0, 0.5, 1, 2, 4, 8, and 20 h. For comparison, the same total amount of DNAJB6 was added at the beginning of the experiment in the (C) unseeded and (D) seeded aggregation reactions.

FIGURE 8.

Extension of lag-phase by sequestering of growing Aβ42 aggregates by DNAJB6. The increase in the half-times of the reaction profiles shown in Fig. 7 as a function of DNAJB6 concentration. The symbol t_0.5* represents the half-time of the aggregation reaction in the absence of chaperone. The linear increase observed for both unseeded and seeded reactions suggests that DNAJB6 binds to the growing aggregates with high affinity as described in the main text.