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The Hsc70 Disaggregation Machinery Removes Monomer Units Directly from α-Synuclein Fibril Ends

View ORCID ProfileMatthias M. Schneider, Saurabh Gautam, View ORCID ProfileTherese W. Herling, Ewa Andrzejewska, View ORCID ProfileGeorg Krainer, Alyssa M. Miller, View ORCID ProfileQuentin A. E. Peter, View ORCID ProfileFrancesco Simone Ruggeri, Michele Vendruscolo, Andreas Bracher, Christopher M. Dobson, F. Ulrich Hartl, Tuomas P. J. Knowles
doi: https://doi.org/10.1101/2020.11.02.365825
Matthias M. Schneider
1Centre for Misfolding Diseases, Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom
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  • ORCID record for Matthias M. Schneider
Saurabh Gautam
2Department of Cellular Biochemistry, Max-Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany
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Therese W. Herling
1Centre for Misfolding Diseases, Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom
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  • ORCID record for Therese W. Herling
Ewa Andrzejewska
1Centre for Misfolding Diseases, Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom
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Georg Krainer
1Centre for Misfolding Diseases, Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom
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Alyssa M. Miller
1Centre for Misfolding Diseases, Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom
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Quentin A. E. Peter
1Centre for Misfolding Diseases, Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom
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  • ORCID record for Quentin A. E. Peter
Francesco Simone Ruggeri
1Centre for Misfolding Diseases, Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom
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  • ORCID record for Francesco Simone Ruggeri
Michele Vendruscolo
1Centre for Misfolding Diseases, Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom
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Andreas Bracher
2Department of Cellular Biochemistry, Max-Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany
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Christopher M. Dobson
1Centre for Misfolding Diseases, Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom
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F. Ulrich Hartl
2Department of Cellular Biochemistry, Max-Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany
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  • For correspondence: uhartl@biochem.mpg.de tpjk2@cam.ac.uk
Tuomas P. J. Knowles
1Centre for Misfolding Diseases, Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom
3Cavendish Laboratory, Department of Physics, University of Cambridge, JJ Thomson Road, Cambridge CB3 0HE, United Kingdom
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  • For correspondence: uhartl@biochem.mpg.de tpjk2@cam.ac.uk
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Abstract

Molecular chaperones contribute to the maintenance of cellular protein homeostasis through a wide range of mechanisms, including the assistance of de novo protein folding, the rescue of misfolded proteins, and the prevention of amyloid formation. Chaperones of the Hsp70 family have a striking capability of disaggregating otherwise irreversible aggregate structures such as amyloid fibrils that accumulate during the development of neurodegenerative diseases. However, the mechanisms of this key emerging functionality remain largely unknown. Here, we bring together microfluidic measurements with kinetic analysis and show that that the Hsp70 protein heat chock complement Hsc70 together with its two co-chaperones DnaJB1 and the nucleotide exchange factor Apg2 is able to completely reverse the aggregation process of alpha-synuclein, associated with Parkinson’s disease, back to its soluble monomeric state. Moreover, we show that this reaction proceeds with first order kinetics in a process where monomer units are taken off directly from the fibril ends. Our results demonstrate that all components of the chaperone triad are essential for fibril disaggregation. Lastly, we quantify the interactions between the three chaperones as well as between the chaperones and the fibrils in solution, yielding both binding stoichiometries and dissociation constants. Crucially, we find that the stoichiometry of Hsc70 binding to fibrils suggests Hsc70 clustering at the fibril ends. Taken together, our results show that the mechanism of action of the Hsc70–DnaJB1–Apg2 chaperone system in disaggregating α-synuclein fibrils involves the removal of monomer units without any intermediate fragmentation steps. These findings are fundamental to our understanding of the suppression of amyloid proliferation early in life and the natural clearance mechanisms of fibrillar deposits in Parkinson’s disease, and inform on the possibilities and limitations of this strategy in the development of therapeutics against synucleinopathies and related neurodegenerative diseases.

Competing Interest Statement

The authors have declared no competing interest.

Footnotes

  • ↵† Deceased (September 2019)

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC 4.0 International license.
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The Hsc70 Disaggregation Machinery Removes Monomer Units Directly from α-Synuclein Fibril Ends
Matthias M. Schneider, Saurabh Gautam, Therese W. Herling, Ewa Andrzejewska, Georg Krainer, Alyssa M. Miller, Quentin A. E. Peter, Francesco Simone Ruggeri, Michele Vendruscolo, Andreas Bracher, Christopher M. Dobson, F. Ulrich Hartl, Tuomas P. J. Knowles
bioRxiv 2020.11.02.365825; doi: https://doi.org/10.1101/2020.11.02.365825
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The Hsc70 Disaggregation Machinery Removes Monomer Units Directly from α-Synuclein Fibril Ends
Matthias M. Schneider, Saurabh Gautam, Therese W. Herling, Ewa Andrzejewska, Georg Krainer, Alyssa M. Miller, Quentin A. E. Peter, Francesco Simone Ruggeri, Michele Vendruscolo, Andreas Bracher, Christopher M. Dobson, F. Ulrich Hartl, Tuomas P. J. Knowles
bioRxiv 2020.11.02.365825; doi: https://doi.org/10.1101/2020.11.02.365825

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