Structural Mechanisms of Mutant Huntingtin Aggregation Suppression by the Synthetic Chaperonin-like CCT5 Complex Explained by Cryoelectron Tomography

Michele C Darrow,Oksana A Sergeeva,Jose M Isas,Jesús G Galaz-Montoya,Jonathan A King,Ralf Langen,Michael F Schmid,Wah Chiu

doi:10.1074/jbc.m115.655373

Abstract

Huntington disease, a neurodegenerative disorder characterized by functional deficits and loss of striatal neurons, is linked to an expanded and unstable CAG trinucleotide repeat in the huntingtin gene (HTT). This DNA sequence translates to a polyglutamine repeat in the protein product, leading to mutant huntingtin (mHTT) protein aggregation. The aggregation of mHTT is inhibited in vitro and in vivo by the TCP-1 ring complex (TRiC) chaperonin. Recently, a novel complex comprised of a single type of TRiC subunit has been reported to inhibit mHTT aggregation. Specifically, the purified CCT5 homo-oligomer complex, when compared with TRiC, has a similar structure, ATP use, and substrate refolding activity, and, importantly, it also inhibits mHTT aggregation. Using an aggregation suppression assay and cryoelectron tomography coupled with a novel computational classification method, we uncover the interactions between the synthetic CCT5 complex (∼ 1 MDa) and aggregates of mutant huntingtin exon 1 containing 46 glutamines (mHTTQ46-Ex1). We find that, in a similar fashion to TRiC, synthetic CCT5 complex caps mHTT fibrils at their tips and encapsulates mHTT oligomers, providing a structural description of the inhibition of mHTTQ46-Ex1 by CCT5 complex and a shared mechanism of mHTT inhibition between TRiC chaperonin and the CCT5 complex: cap and contain.

Highlights

Huntington disease patients show an accumulation of oligomers and fibrillar species of mutant huntingtin
Using an aggregation suppression assay and cryoelectron tomography coupled with a novel computational classification method, we uncover the interactions between the synthetic CCT5 complex (ϳ1 MDa) and aggregates of mutant huntingtin exon 1 containing 46 glutamines
Using a cleavable linker on a construct containing mutant huntingtin (mHTT) with 46 polyglutamines allowed us to follow the process of mHTT aggregation over time in the absence and presence of the CCT5 complex

Summary

Background

Huntington disease patients show an accumulation of oligomers and fibrillar species of mutant huntingtin (mHTT). Huntington disease, a neurodegenerative disorder characterized by functional deficits and loss of striatal neurons, is linked to an expanded and unstable CAG trinucleotide repeat in the huntingtin gene (HTT) This DNA sequence translates to a polyglutamine repeat in the protein product, leading to mutant huntingtin (mHTT) protein aggregation. Aggregates of mutant huntingtin (mHTT) protein have been identified in mouse models and found in patient brains [4, 5] These aggregates contain fragments of mHTT protein, including those composed of only the first exon of Htt, the region where the polyglutamine repeat is located [6]. The CCT5 complex forms a double-ring barrel structure composed of 16 identical CCT5 subunits [23] This chaperonin-like homo-oligomeric complex hydrolyzes ATP and refolds both luciferase and human ␥-Dcrystallin [23]. It has been reported recently that the CCT5 complex inhibits mHTT aggregation [24], leading us to question whether the CCT5 complex utilizes the cap and contain structural mechanisms to inhibit mHTT aggregation

Experimental Procedures

Results

Discussion