The deposition of highly ordered fibrillar-type aggregates into inclusion bodies is a hallmark of neurodegenerative diseases such as Parkinson’s disease. The high stability of such amyloid fibril aggregates makes them challenging substrates for the cellular protein quality-control machinery1,2. However, the human HSP70 chaperone and its co-chaperones DNAJB1 and HSP110 can dissolve preformed fibrils of the Parkinson’s disease-linked presynaptic protein α-synuclein in vitro3,4. The underlying mechanisms of this unique activity remain poorly understood. Here we use biochemical tools and nuclear magnetic resonance spectroscopy to determine the crucial steps of the disaggregation process of amyloid fibrils. We find that DNAJB1 specifically recognizes the oligomeric form of α-synuclein via multivalent interactions, and selectively targets HSP70 to fibrils. HSP70 and DNAJB1 interact with the fibril through exposed, flexible amino and carboxy termini of α-synuclein rather than the amyloid core itself. The synergistic action of DNAJB1 and HSP110 strongly accelerates disaggregation by facilitating the loading of several HSP70 molecules in a densely packed arrangement at the fibril surface, which is ideal for the generation of ‘entropic pulling’ forces. The cooperation of DNAJB1 and HSP110 in amyloid disaggregation goes beyond the classical substrate targeting and recycling functions that are attributed to these HSP70 co-chaperones and constitutes an active and essential contribution to the remodelling of the amyloid substrate. These mechanistic insights into the essential prerequisites for amyloid disaggregation may provide a basis for new therapeutic interventions in neurodegeneration.
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All data presented in this Article are available within the figures and Supplementary Information files. All other data are available from the corresponding authors upon reasonable request. Source data are provided with this paper.
Custom fortran90 code for kinetic Monte Carlo simulations of the effect of crowding on HSP70 binding to amyloid fibrils is available from https://github.com/a-barducci/Hsp70crowding-kMC.
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We thank K. Chi and B. Simon for experimental assistance and A. Mogk for critical reading of the manuscript. N.B.N. is supported by Recruitment Grant from FMNHS, Monash University with funding from Victorian and Australian Governments. J.H. gratefully acknowledges the European Molecular Biology Laboratory (EMBL) for supporting this work. This work was financed by grants from the Baden-Württemberg Stiftung (BWST-INTSFIII-029), the Helmholtzgemeinschaft (AMPro) and the Deutsche Forschungsgemeinschaft (DFG) (project 201348542 – SFB 1036 and BU617/19-1) to B.B.
The authors declare no competing interests.
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Extended data figures and tables
a–d, Cartoon representations of DNAJB134(a), HSP70 (HSC70 homology model generated by SWISS-MODEL39 from PDB 2KHO40) (b), HSP110 (HSPH2 homology model generated by SWISS-MODEL from PDB 2QXL41) (c) and α-syn amyloid filament (PDB 2N0A42) (d), all rendered to scale to illustrate the relatively large size of the HSP70 machine compared to the fibrillar substrate. Boundaries to the amyloid core are indicated by their residue number. CTD, C-terminal domain; DD, dimerization domain; JD, J-domain; NBD, nucleotide-binding domain; SBD, substrate-binding domain.
a–e, α-synuclein (100 μM) 1H–15N HSQC signal intensity ratios (Ichap/I0) after the addition of 50 μM HSP110 (a), 250 μM DNAJB1+ 500 μM HSP70 (b), 250 μM DNAJB1 + 50 μM HSP110 (c), 500 μM HSP70 + 50 μM HSP110 (d) and 250 μM DNAJB1 + 500 μM HSP70 + 50 μM HSP110 (e). Low intensity ratios reflect chaperone binding, illustrated in the cartoon representations of the α-syn sequence. Protein concentrations were chosen to reflect chaperone ratios in which maximum disaggregation activity is detected. The NAC region is indicated with grey dashes.
Extended Data Fig. 3 C-terminal truncation abolishes DNAJB1 interaction and disaggregation activity.
a, b, Cartoon representations of an α-syn amyloid protofilament (PDB 2N0A42) illustrating the effects of chaperone binding (a, DNAJB1; b, HSP70) on site specific BADAN fluorescence (Fig. 1c, d) introduced at the indicated sites. The colour gradient reflects the strength of the interaction. DNAJB1 binding primarily affects fluorophores introduced at the C terminus, whereas the effects of HSP70 binding are specific to the N terminus. c, Schematic representation of N- and C-terminal truncation constructs of α-syn. d, Immunoblot of insoluble (pellet, P) and soluble (S) fractions of fibrillar α-syn truncation mutants in the absence or presence of chaperones (DNAJB1, HSP70, HSP110) and ATP after 16-h incubation. e, Steady-state fluorescence anisotropy of DNAJB1AF488 after titration of indicated fibrillar C-terminal truncations of α-syn. Binding curves were fitted to a one-site binding model using GraphPad Prism. f, Disaggregation of fibrillar C-terminal truncations of α-syn by the HSP70 chaperone machinery (DNAJB1, HSP70, HSP110) and ATP monitored by ThT fluorescence. Reaction conditions and number of independent replicates are specified in Extended Data Table 2. Data are mean ± s.e.m.
a, Kinetics of DNAJB1AF488 dissociation from α-syn fibrils after competition with 100× excess unlabelled wild-type DNAJB1 in the presence of 2× HSP70 and/or 0.2× HSP110. JB1 dissociation rates in the absence of additional chaperones (from Fig. 2d) are included as reference. b, Titration of HSP70AF488 with α-syn fibrils in the absence (black) or presence (orange) of 0.5× DNAJB1 determined by steady-state fluorescence anisotropy. c, Steady-state fluorescence anisotropy of HSP70AF488 after the addition of fibrillar α-syn truncation mutants in the presence of 0.5× DNAJB1. Anisotropy measurements in the absence of DNAJB1 (from Fig. 1g) are included as reference. d, e, Stoichiometry of chaperone binding to α-syn fibrils. Quantification of the co-sedimentation of HSP70 in the presence of DNAJB1 (d) or DNAJB1 alone (e) with 20 μM α-syn fibrils under saturating conditions as a function of chaperone concentration. Linear regression of non-saturated and saturated data points (dashed lines) reveals a stoichiometry of 1:10 for the DNAJB1 dimer and 1:2 for HSP70 in the presence of DNAJB1. Source data are provided in Supplementary Fig. 1. Reaction conditions and number of replicates are specified in Extended Data Table 2. Data are mean ± s.e.m. Solid lines represent fits of the data to the quadratic solution of the equilibrium binding equation.
a, Entropic pulling: HSP70 bound to monomeric α-syn can freely move in all directions. Fibril bound HSP70 in contrast collides with the amyloid surface. The extent to which its movement is restricted depends on how close to the amyloid surface the chaperone is bound. Frequent collision with the amyloid surface generates a momentum away from the amyloid surface, pulling the bound α-syn protomer along. This model of HSP70 chaperone action is termed entropic pulling. Entropic-pulling forces can be amplified by a high overall density of chaperones, which results in collisions not only with the fibril surface but also between chaperones. b, HSP70 oligomerization. Fluorescence intensity of HSP70AF488 after titration with HSP70AF594. Data are mean ± s.e.m. c, Apparent HSP70 dissociation constants for α-syn fibrils in the presence of DNAJB1, derived from the titration data in Fig. 3b by fit of the mean polarization values at each concentration of fibrils to a binding model assuming a 1:1 (black) or 1:2 (orange) HSP70 to α-syn stoichiometry. Reaction conditions and number of replicates are specified in Extended Data Table 2.
a, Disaggregation of α-syn fibrils by the HSP70 machinery (DNAJB1, HSP70) in the presence of wild-type HSP110 (black), ATPase-deficient HSP110 (light green, D7S)23 or NEF-deficient (ΔNEF) HSP110 (green, A279A/N280A/N619Y/E622A)23. b, Dissociation of HSP70AF488 from amyloid fibrils triggered by the addition of 0.1× wild-type HSP110 (black), ATPase deficient HSP110 (light green) or NEF-deficient HSP110 (green) in the presence of ATP. c, Steady-state ATPase rates of HSP70 in the presence of 1× DNAJB1 and 2× BAG1 (red), 2× H6-sumo-BAG1 (orange) or 2× H6-sumo-BAG1 with 2.5× anti-H6 (green), data are mean ± s.e.m. d, ThT fluorescence-based disaggregation assay of α-syn fibrils with the full (untagged) HSP70 machinery (DNAJB1, HSP70, HSP110) in the absence (black) or presence of 1.25× anti-H6 (red). Reaction conditions and number of replicates are specified in Extended Data Table 2.
a, Disaggregation of α-syn fibrils monitored by ThT fluorescence. Preformed fibrils of α-syn were incubated with DNAJB1, 1× HSP70 or 5× HSP70 and in the absence or presence of HSP110 as indicated. b, Fold change in FRET efficiencies (A/D) between equimolar HSP70AF488 and HSP70AF594 in presence of 20× α-syn fibrils, 0.5× DNAJB1 and ATP after titration of HSP110 (2.5, 5, 10, 20, 40, 80, 160, 320, 640 nM) relative to the absence of NEF. c, Steady-state HSP70AF488 polarization in the presence of 0.5× DNAJB1 and 20× α-syn fibrils as a function of HSP110 concentration. d, Fold change in FRET efficiencies (A/D) of indicated HSP70 donor (AF488) and acceptor (AF594) pairs (numbering indicates position of modified cysteine residue in HSP70) in presence of 20× α-syn fibrils, 0.5× DNAJB1 and ATP after titration of HSP110 (8, 25, 74, 222, 667 nM, 2, 6 μM) compared to the absence of NEF. e, Fold change in HSP70 FRET efficiencies (A/D in presence of 20× α-syn fibrils, 0.5× DNAJB1 and ATP after titration of HSP110 (2.5, 5, 10, 20, 40, 80, 160, 320, 640 nM) or BAG1 constructs (12.5, 25, 50, 100, 200, 400, 800 nM, 1.6, 3.2 μM) relative to the absence of NEF. Reaction conditions and number of independent replicates are specified in Extended Data Table 2. For all plots, data are mean ± s.e.m. Solid lines represent fits of the data to the one-site binding equation.
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Wentink, A.S., Nillegoda, N.B., Feufel, J. et al. Molecular dissection of amyloid disaggregation by human HSP70. Nature 587, 483–488 (2020). https://doi.org/10.1038/s41586-020-2904-6
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