Stable α-Synuclein Oligomers Strongly Inhibit Chaperone Activity of the Hsp70 System by Weak Interactions with J-domain Co-chaperones

Pierre Goloubinoff,Hilal A Lashuel,Marie-Pierre Hinault,America Farina Henriquez Cuendet,Mounir Mensi,Rayees U.H Mattoo,Giovanni Dietler

doi:10.1074/jbc.m110.127753

Abstract

α-Synuclein aggregation and accumulation in Lewy bodies are implicated in progressive loss of dopaminergic neurons in Parkinson disease and related disorders. In neurons, the Hsp70s and their Hsp40-like J-domain co-chaperones are the only known components of chaperone network that can use ATP to convert cytotoxic protein aggregates into harmless natively refolded polypeptides. Here we developed a protocol for preparing a homogeneous population of highly stable β-sheet enriched toroid-shaped α-Syn oligomers with a diameter typical of toxic pore-forming oligomers. These oligomers were partially resistant to in vitro unfolding by the bacterial Hsp70 chaperone system (DnaK, DnaJ, GrpE). Moreover, both bacterial and human Hsp70/Hsp40 unfolding/refolding activities of model chaperone substrates were strongly inhibited by the oligomers but, remarkably, not by unstructured α-Syn monomers even in large excess. The oligomers acted as a specific competitive inhibitor of the J-domain co-chaperones, indicating that J-domain co-chaperones may preferably bind to exposed bulky misfolded structures in misfolded proteins and, thus, complement Hsp70s that bind to extended segments. Together, our findings suggest that inhibition of the Hsp70/Hsp40 chaperone system by α-Syn oligomers may contribute to the disruption of protein homeostasis in dopaminergic neurons, leading to apoptosis and tissue loss in Parkinson disease and related neurodegenerative diseases.

Highlights

A number of neurodegenerative disorders, such as Alzheimer, amyotrophic lateral sclerosis, Huntington, prion encephalopathy, and Parkinson diseases (PDs)2 are characterized by a loss of neurons associated with protein misfolding and the accumulation in and outside cells of stable protein aggregates composed of specific proteins, such as tau tangles, amyloid-␤ plaques, ␣-Syn fibrils, and Lewy bodies [1]
We obtained a much reduced fraction, albeit of ϳ95% pure ␣-Syn, that was devoid of large insoluble species, composed of fully soluble, discrete oligomers that resolved on native gel mainly as 10 –14 mers (140 –200 kDa) as well as some dimers and monomers (Fig. 1B, lane 3)
As expected from the native gel (Fig. 1B), we observed two broad peaks; one corresponding to soluble high molecular weight (Mr) ␣-Syn oligomers ranging from 102 to 103 kDa (7.5–11 ml) with a very high specific affinity for the amyloid specific dye, thioflavin T (Th-T), and a second that was resolved at the expected position of the 14.5-kDa ␣-Syn monomer (15–18 ml) with a very low specific affinity for Th-T (Fig. 2A, dark line)

Summary

Introduction

A number of neurodegenerative disorders, such as Alzheimer, amyotrophic lateral sclerosis, Huntington, prion encephalopathy, and Parkinson diseases (PDs)2 are characterized by a loss of neurons associated with protein misfolding and the accumulation in and outside cells of stable protein aggregates composed of specific proteins, such as tau tangles, amyloid-␤ plaques, ␣-Syn fibrils, and Lewy bodies [1]. Both bacterial and human Hsp70/Hsp40 unfolding/refolding activities of model chaperone substrates were strongly inhibited by the oligomers but, remarkably, not by unstructured ␣-Syn monomers even in large excess.

Results

Conclusion