Hsp70 and Hsp40 Functionally Interact with Soluble Mutant Huntingtin Oligomers in a Classic ATP-dependent Reaction Cycle

Gregor P Lotz,Justin Legleiter,Rebecca Aron,Emily J Mitchell,Shao-Yi Huang,Cheping Ng,Charles Glabe,Leslie M Thompson,Paul J Muchowski

doi:10.1074/jbc.m110.160218

Abstract

Inclusion bodies of aggregated mutant huntingtin (htt) fragments are a neuropathological hallmark of Huntington disease (HD). The molecular chaperones Hsp70 and Hsp40 colocalize to inclusion bodies and are neuroprotective in HD animal models. How these chaperones suppress mutant htt toxicity is unclear but might involve direct effects on mutant htt misfolding and aggregation. Using size exclusion chromatography and atomic force microscopy, we found that mutant htt fragments assemble into soluble oligomeric species with a broad size distribution, some of which reacted with the conformation-specific antibody A11. Hsp70 associated with A11-reactive oligomers in an Hsp40- and ATP-dependent manner and inhibited their formation coincident with suppression of caspase 3 activity in PC12 cells. Thus, Hsp70 and Hsp40 (DNAJB1) dynamically target specific subsets of soluble oligomers in a classic ATP-dependent reaction cycle, supporting a pathogenic role for these structures in HD.

Highlights

Inclusion bodies of aggregated mutant huntingtin fragments are a neuropathological hallmark of Huntington disease (HD)
Using size exclusion chromatography and atomic force microscopy, we found that mutant htt fragments assemble into soluble oligomeric species with a broad size distribution, some of which reacted with the conformation-specific antibody A11
Anti-oligomer Antibody A11 Does Not React with Full-length Hsp70—Previously, we showed by atomic force microscopy (AFM) that Hsp70 and Hsp40 suppress formation of mutant htt oligomers but do not destabilize preformed oligomers [7], suggesting that these chaperones act on mutant htt monomers or possibly early soluble oligomers

Summary

EXPERIMENTAL PROCEDURES

Protein Purification—GST-HD20Q and GST-HD53Q were purified as described [7, 15, 24]. Immediately after SEC, fresh, unfrozen HD53Q and HD20Q were used for all experiments. For dot-blot analyses, equal amounts of protein (2 ␮g) were removed from 6 ␮M aggregation reactions at various times after GST cleavage and applied to a nitrocellulose membrane (0.2-␮m pore size; Schleicher & Schuell). For Western blot analyses, proteins and aggregates were separated by SDS-PAGE and transferred to nitrocellulose membranes (0.45-␮m pore size; Schleicher & Schuell) by standard Western transfer techniques and treated as described [7]. 38184 JOURNAL OF BIOLOGICAL CHEMISTRY were collected and applied to a nitrocellulose membrane (0.1-␮m pore size; Schleicher & Schuell) through a slot blot manifold (Hoefer PR 648) without any washing steps or detergent treatment Both soluble monomeric and aggregated forms of HD53Q were retained in a nondenatured state on this charged, strong protein-binding membrane and analyzed with the antibodies as indicated. Significant differences (*, p Ͻ 0.05; **, p Ͻ 0.01; and ***, p Ͻ 0.001) are marked with asterisks

RESULTS

Mutant htt Oligomers Inhibit

DISCUSSION