The Molecular Chaperone Hsp90 Modulates Intermediate Steps of Amyloid Assembly of the Parkinson-related Protein α-Synuclein

S.Fabio Falsone,Andreas J Kungl,Angelika Rek,Roberto Cappai,Klaus Zangger

doi:10.1074/jbc.m109.057240

S.Fabio Falsone, Andreas J Kungl + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m109.057240

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Abstract

Alpha-synuclein is an intrinsically unstructured protein that binds to membranes, forms fibrils, and is involved in neurodegeneration. We used a reconstituted in vitro system to show that the molecular chaperone Hsp90 influenced alpha-synuclein vesicle binding and amyloid fibril formation, two processes that are tightly coupled to alpha-synuclein folding. Binding of Hsp90 to monomeric alpha-synuclein occurred in the low micromolar range, involving regions of alpha-synuclein that are critical for vesicle binding and amyloidogenesis. As a consequence, both processes were affected. In the absence of ATP, the accumulation of non-amyloid alpha-synuclein oligomers prevailed over fibril formation, whereas ATP favored fibril growth. This suggests that Hsp90 modulates the assembly of alpha-synuclein in an ATP-dependent manner. We propose that Hsp90 affects these folding processes by restricting conformational fluctuations of alpha-synuclein.

Highlights

The physiological function of AS is still elusive [3, 4]
Our analysis reveals that Hsp90 binds to AS and abolishes binding of this polypeptide to small unilamellar vesicles
ATP Drives Hsp90-mediated AS Assembly—we studied the influence of Hsp90 on AS fibril formation

Summary

EXPERIMENTAL PROCEDURES

All reagents were from Sigma. Protein Expression and Purification—The expression vector containing the N-terminal His6-fused HSP90␤ gene (a kind gift of Sophie Jackson, Cambridge, UK) was transformed into Bl21DE3(Star) cells (Invitrogen). The protein was dialyzed against 20 mM Tris, 50 mM NaCl, 5 mM dithiothreitol, pH 7.4, and loaded on a Resource Q column (GE Healthcare). The protein was purified according to Ref. 22 and extensively dialyzed against 50 mM NH4HCO3 Both unlabeled and labeled AS were aliquoted, lyophilized, and stored at Ϫ80 °C. Fluorescence Titrations—Fluorescence emission spectra of a 100 nM Hsp or Hsp solution in 20 mM Tris-HCl, 150 mM NaCl, pH 7.4, were measured using a LB50 spectrofluorimeter (PerkinElmer Life Sciences) at 25 °C and an excitation wavelength of 295 nm. Fluorescence Anisotropy—The fluorescence anisotropy of a 1 ␮M DPH-labeled SUV preparation in 20 mM Na2HPO4, pH 7.4, was measured at 25 °C using a LB50 spectrofluorimeter (PerkinElmer Life Sciences) at an emission wavelength of 430 nm upon excitation at 359 nm.

The reaction was monitored by the

RESULTS

DISCUSSION

Stokes radius r