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

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.

Hsp70 chaperone blocks α-synuclein oligomer formation via a novel engagement mechanism

“…Hsp/c70s have two known mechanisms of mitigating protein misfolding: an ATPcycling dependent "foldase" action, which is able to restore damaged proteins, and an ATP-independent "holdase" mode, which binds to unfolded proteins to prevent aggregation (23). Although in concert with cochaperones Hsp70 can disaggregate ASyn fibrils in an ATP-cycling mode (24,25), Hsp/c70s block ASyn fibrillization (26)(27)(28)(29)(30)(31)(32)(33) in an ATP-independent manner (27)(28)(29)(30)(31)(32), indicating a "holdase"-based mechanism. It has been assumed that this holdase-like activity was mediated via Hsp/ c70's canonical substrate-binding site in the SBD, which is required for other known holdase roles (34).…”

Chaperones Skp and SurA dynamically expand unfolded outer membrane protein X and synergistically disassemble oligomeric aggregates

“…Surprisingly, we found that Skp 3 and SurA act synergistically on uOmpX aggregates, suggesting higher order interactions between both periplasmic chaperones. Disaggregation mechanisms have also been observed in the ATP-dependent cytoplasmic chaperones of Hsp70 family 68,69 . However, both Skp and SurA disaggregate oligomeric structures in the absence of an external energy source, thereby supporting the known disaggregase DegP, with a yet to be determined mechanism.…”

LAG3 is not expressed in human and murine neurons and does not modulate α-synucleinopathies

“…We therefore speculated that surface effects may lead to electrostatic interactions. We therefore employed a microfluidics-based technology (Arosio et al, 2016;Scheidt et al, 2019;Schneider et al, 2020) to characterize the receptor ligand interaction via diffusional sizing.…”

“…The samples were incubated at room temperature for 60 minutes and the size of the formed immunocomplex was determined through measuring the hydrodynamic radius, Rh, with microfluidic diffusional sizing, as described above. Dissociation constants (KD) were determined using the Langmuir binding isotherm, as previously reported (Schneider et al, 2020),…”