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Molecular dissection of amyloid disaggregation by human HSP70

An Author Correction to this article was published on 22 December 2020

This article has been updated

Abstract

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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Fig. 1: The HSP70 machinery binds to discrete sequences in α-synuclein.
Fig. 2: Specific interaction of DNAJB1 with α-syn fibrils leads to clustering of HSP70 molecules.
Fig. 3: DNAJB1 facilitates HSP70 binding at high density to α-syn fibrils.
Fig. 4: HSP110 potentiates amyloid disaggregation.
Fig. 5: Model of amyloid disaggregation by the HSP70 chaperone machinery.

Data availability

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.

Code availability

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.

Change history

  • 22 December 2020

    A Correction to this paper has been published: https://doi.org/10.1038/s41586-020-03090-x.

References

  1. Wentink, A., Nussbaum-Krammer, C. & Bukau, B. Modulation of amyloid states by molecular chaperones. Cold Spring Harb. Perspect. Biol. 11, a033969 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Kampinga, H. H. & Bergink, S. Heat shock proteins as potential targets for protective strategies in neurodegeneration. Lancet Neurol. 15, 748–759 (2016).

    CAS  PubMed  Google Scholar 

  3. Duennwald, M. L., Echeverria, A. & Shorter, J. Small heat shock proteins potentiate amyloid dissolution by protein disaggregases from yeast and humans. PLoS Biol. 10, e1001346 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Gao, X. et al. Human Hsp70 disaggregase reverses Parkinson’s-linked α-synuclein amyloid fibrils. Mol. Cell 59, 781–793 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Rosenzweig, R., Nillegoda, N. B., Mayer, M. P. & Bukau, B. The Hsp70 chaperone network. Nat. Rev. Mol. Cell Biol. 20, 665–680 (2019).

    CAS  PubMed  Google Scholar 

  6. Mayer, M. P. & Gierasch, L. M. Recent advances in the structural and mechanistic aspects of Hsp70 molecular chaperones. J. Biol. Chem. 294, 2085–2097 (2019).

    CAS  PubMed  Google Scholar 

  7. Bracher, A. & Verghese, J. The nucleotide exchange factors of Hsp70 molecular chaperones. Front. Mol. Biosci. 2, 10 (2015).

    PubMed  PubMed Central  Google Scholar 

  8. Nachman, E. et al. Disassembly of Tau fibrils by the human Hsp70 disaggregation machinery generates small seeding-competent species. J. Biol. Chem. 295, 9676–9690 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Scior, A. et al. Complete suppression of Htt fibrilization and disaggregation of Htt fibrils by a trimeric chaperone complex. EMBO J. 37, 282–299 (2018).

    CAS  PubMed  Google Scholar 

  10. Burmann, B. M. et al. Regulation of α-synuclein by chaperones in mammalian cells. Nature 577, 127–132 (2019).

    PubMed  PubMed Central  Google Scholar 

  11. Redeker, V., Pemberton, S., Bienvenut, W., Bousset, L. & Melki, R. Identification of protein interfaces between α-synuclein, the principal component of Lewy bodies in Parkinson disease, and the molecular chaperones human Hsc70 and the yeast Ssa1p. J. Biol. Chem. 287, 32630–32639 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Uéda, K. et al. Molecular cloning of cDNA encoding an unrecognized component of amyloid in Alzheimer disease. Proc. Natl Acad. Sci. USA 90, 11282–11286 (1993).

    ADS  PubMed  PubMed Central  Google Scholar 

  13. Guerrero-Ferreira, R., Kovacik, L., Ni, D. & Stahlberg, H. New insights on the structure of alpha-synuclein fibrils using cryo-electron microscopy. Curr. Opin. Neurobiol. 61, 89–95 (2020).

    CAS  PubMed  Google Scholar 

  14. Vilar, M. et al. The fold of alpha-synuclein fibrils. Proc. Natl Acad. Sci. USA 105, 8637–8642 (2008).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  15. Sha, B., Lee, S. & Cyr, D. M. The crystal structure of the peptide-binding fragment from the yeast Hsp40 protein Sis1. Structure 8, 799–807 (2000).

    CAS  PubMed  Google Scholar 

  16. Jiang, Y., Rossi, P. & Kalodimos, C. G. Structural basis for client recognition and activity of Hsp40 chaperones. Science 365, 1313–1319 (2019).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  17. De Los Rios, P. & Barducci, A. Hsp70 chaperones are non-equilibrium machines that achieve ultra-affinity by energy consumption. eLife 3, e02218 (2014).

    PubMed  PubMed Central  Google Scholar 

  18. Lu, Z. & Cyr, D. M. Protein folding activity of Hsp70 is modified differentially by the Hsp40 co-chaperones Sis1 and Ydj1. J. Biol. Chem. 273, 27824–27830 (1998).

    CAS  PubMed  Google Scholar 

  19. De Los Rios, P., Ben-Zvi, A., Slutsky, O., Azem, A. & Goloubinoff, P. Hsp70 chaperones accelerate protein translocation and the unfolding of stable protein aggregates by entropic pulling. Proc. Natl Acad. Sci. USA 103, 6166–6171 (2006).

    ADS  PubMed  PubMed Central  Google Scholar 

  20. Goloubinoff, P. & De Los Rios, P. The mechanism of Hsp70 chaperones: (entropic) pulling the models together. Trends Biochem. Sci. 32, 372–380 (2007).

    CAS  PubMed  Google Scholar 

  21. Sousa, R. & Lafer, E. M. The physics of entropic pulling: a novel model for the Hsp70 motor mechanism. Int. J. Mol. Sci. 20, E2334 (2019).

    PubMed  Google Scholar 

  22. Sousa, R. et al. Clathrin-coat disassembly illuminates the mechanisms of Hsp70 force generation. Nat. Struct. Mol. Biol. 23, 821–829 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Rampelt, H. et al. Metazoan Hsp70 machines use Hsp110 to power protein disaggregation. EMBO J. 31, 4221–4235 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Easton, D. P., Kaneko, Y. & Subjeck, J. R. The hsp110 and Grp1 70 stress proteins: newly recognized relatives of the Hsp70s. Cell Stress Chaperones 5, 276–290 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Faust, A. O. et al. HSP40s use class-specific regulation to drive HSP70 functional diversity. Nature https://doi.org/10.1038/s41586-020-2906-4 (2020).

  26. Assenza, S. et al. Efficient conversion of chemical energy into mechanical work by Hsp70 chaperones. eLife 8, e48491 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Imamoglu, R., Balchin, D., Hayer-Hartl, M. & Hartl, F. U. Bacterial Hsp70 resolves misfolded states and accelerates productive folding of a multi-domain protein. Nat. Commun. 11, 365 (2020).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  28. Kellner, R. et al. Single-molecule spectroscopy reveals chaperone-mediated expansion of substrate protein. Proc. Natl Acad. Sci. USA 111, 13355–13360 (2014).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  29. Sharma, S. K., De los Rios, P., Christen, P., Lustig, A. & Goloubinoff, P. The kinetic parameters and energy cost of the Hsp70 chaperone as a polypeptide unfoldase. Nat. Chem. Biol. 6, 914–920 (2010).

    CAS  PubMed  Google Scholar 

  30. Kmiecik, S. W., Le Breton, L. & Mayer, M. P. Feedback regulation of heat shock factor 1 (Hsf1) activity by Hsp70-mediated trimer unzipping and dissociation from DNA. EMBO J. 39, e104096 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Pieri, L., Madiona, K., Bousset, L. & Melki, R. Fibrillar α-synuclein and huntingtin exon 1 assemblies are toxic to the cells. Biophys. J. 102, 2894–2905 (2012).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  32. Taguchi, Y. V. et al. Hsp110 mitigates α-synuclein pathology in vivo. Proc. Natl Acad. Sci. USA 116, 24310–24316 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Tittelmeier, J. et al. The HSP110/HSP70 disaggregation system generates spreading-competent toxic α-synuclein species. EMBO J. 39, e103954 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Nillegoda, N. B. et al. Crucial HSP70 co-chaperone complex unlocks metazoan protein disaggregation. Nature 524, 247–251 (2015).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  35. Paleologou, K. E. et al. Phosphorylation at Ser-129 but not the phosphomimics S129E/D inhibits the fibrillation of alpha-synuclein. J. Biol. Chem. 283, 16895–16905 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Hoyer, W. et al. Dependence of α-synuclein aggregate morphology on solution conditions. J. Mol. Biol. 322, 383–393 (2002).

    CAS  PubMed  Google Scholar 

  37. Delaglio, F. et al. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6, 277–293 (1995).

    CAS  PubMed  Google Scholar 

  38. Lee, W., Tonelli, M. & Markley, J. L. NMRFAM-SPARKY: enhanced software for biomolecular NMR spectroscopy. Bioinformatics 31, 1325–1327 (2015).

    PubMed  Google Scholar 

  39. Waterhouse, A. et al. SWISS-MODEL: homology modelling of protein structures and complexes. Nucleic Acids Res. 46, W296–W303 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Bertelsen, E. B., Chang, L., Gestwicki, J. E. & Zuiderweg, E. R. Solution conformation of wild-type E. coli Hsp70 (DnaK) chaperone complexed with ADP and substrate. Proc. Natl Acad. Sci. USA 106, 8471–8476 (2009).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  41. Liu, Q. & Hendrickson, W. A. Insights into Hsp70 chaperone activity from a crystal structure of the yeast Hsp110 Sse1. Cell 131, 106–120 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Tuttle, M. D. et al. Solid-state NMR structure of a pathogenic fibril of full-length human α-synuclein. Nat. Struct. Mol. Biol. 23, 409–415 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

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.

Author information

Authors and Affiliations

Authors

Contributions

A.S.W. and B.B. conceived the study. A.S.W., N.B.N., P.D.L.R., A.B., J.H. and B.B. designed experiments. A.S.W., J.F., G.U., N.B.N. and C.P.S. performed biochemical experiments and data analysis. A.S.W. and J.H. performed NMR spectroscopy experiments. A.B. and P.D.L.R. performed numerical simulations. A.S.W., N.B.N., P.D.L.R., A.B., J.H. and B.B. wrote the manuscript.

Corresponding authors

Correspondence to Anne S. Wentink or Bernd Bukau.

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The authors declare no competing interests.

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Peer review information Nature thanks the anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Structural features of the HSP70 disaggregase machinery.

ad, 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.

Extended Data Fig. 2 Interaction of chaperones with monomeric α-syn.

ae, 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.

Source data

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.

Source data

Extended Data Fig. 4 DNAJB1 promotes HSP70 recruitment to α-syn fibrils.

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.

Source data

Extended Data Fig. 5 Entropic-pulling model of amyloid disaggregation by HSP70.

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.

Source data

Extended Data Fig. 6 HSP110 role in disaggregation is independent of ATPase cycle.

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.

Source data

Extended Data Fig. 7 HSP110 role beyond HSP70 recycling.

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.

Source data

Extended Data Table 1 Reaction conditions and replicate numbers for experiments shown in the figures
Extended Data Table 2 Reaction conditions and replicate numbers for experiments shown in the Extended Data figures

Supplementary information

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Supplementary Figure 1

Uncropped immunoblots and gels of the experiments shown in Extended Data Fig. 3d and Extended Data Figure 5d and e.

Supplementary Figure 2

Full 1H-15N HSQC spectra overlays corresponding to the experiments shown in Fig. 1b-d and Extended Data Fig. 2a-e.

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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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