Abstract

Overexpression and aggregation of α-synuclein (ASyn) are linked to the onset and pathology of Parkinson’s disease and related synucleinopathies. Elevated levels of the stress-induced chaperone Hsp70 protect against ASyn misfolding and ASyn-driven neurodegeneration in cell and animal models, yet there is minimal mechanistic understanding of this important protective pathway. It is generally assumed that Hsp70 binds to ASyn using its canonical and promiscuous substrate-binding cleft to limit aggregation. Here we report that this activity is due to a novel and unexpected mode of Hsp70 action, involving neither ATP nor the typical substrate-binding cleft. We use novel ASyn oligomerization assays to show that Hsp70 directly blocks ASyn oligomerization, an early event in ASyn misfolding. Using truncations, mutations, and inhibitors, we confirm that Hsp70 interacts with ASyn via an as yet unidentified, noncanonical interaction site in the C-terminal domain. Finally, we report a biological role for a similar mode of action in H4 neuroglioma cells. Together, these findings suggest that new chemical approaches will be required to target the Hsp70-ASyn interaction in synucleinopathies. Such approaches are likely to be more specific than targeting Hsp70’s canonical action. Additionally, these results raise the question of whether other misfolded proteins might also engage Hsp70 via the same noncanonical mechanism.

Highlights

  • Associated fibril formation [5,6,7,8,9]

  • Purified aggregation of α-synuclein (ASyn) species fused at its C-terminus with either the large (LgBit) or the small (SmBit) portion of NanoBit luciferase (nLuc) were incubated together, and the formation of oligomers was monitored by reconstituted nLuc activity

  • Split nLuc tags placed on separate ASyn molecules reconstituted luciferase activity, whereas removal of ASyn from one of the tags gave minimal background signal (Fig. 1A)

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Summary

Introduction

Associated fibril formation [5,6,7,8,9]. Significant evidence indicates that misfolded ASyn seeding and spread throughout the brain underlie disease progression [10, 11]. To test that our complementation tags do not interfere with fibril seeding ability, we took samples from our nLuc oligomerization assay at different time points and added them to an excess of untagged monomeric ASyn. This mixture was incubated with shaking at 37 C and fibril formation monitored with ThT fluorescence.

Results
Conclusion
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