Co-chaperone CHIP Associates with Expanded Polyglutamine Protein and Promotes Their Degradation by Proteasomes

Keiji Tanaka,Svetlana Kotliarova,Anand Goswami,Nihar Ranjan Jana,Nobuyuki Nukina,Priyanka Dikshit,Shigeo Murata

doi:10.1074/jbc.m412042200

Abstract

A major hallmark of the polyglutamine diseases is the formation of neuronal intranuclear inclusions of the disease proteins that are ubiquitinated and often associated with various chaperones and proteasome components. But, how the polyglutamine proteins are ubiquitinated and degraded by the proteasomes are not known. Here, we demonstrate that CHIP (C terminus of Hsp70-interacting protein) co-immunoprecipitates with the polyglutamine-expanded huntingtin or ataxin-3 and associates with their aggregates. Transient overexpression of CHIP increases the ubiquitination and the rate of degradation of polyglutamine-expanded huntingtin or ataxin-3. Finally, we show that overexpression of CHIP suppresses the aggregation and cell death mediated by expanded polyglutamine proteins and the suppressive effect is more prominent when CHIP is overexpressed along with Hsc70.

Highlights

A major hallmark of the polyglutamine diseases is the formation of neuronal intranuclear inclusions of the disease proteins that are ubiquitinated and often associated with various chaperones and proteasome components
The most likely hypothesis is that the expanded polyglutamine proteins are misfolded, and failure to refold might cause their ubiquitination before they are degraded by proteasome
We identified CHIP ubiquitin ligase that is responsible for the misfolding-dependent ubiquitination of the expanded polyglutamine proteins

Summary

EXPERIMENTAL PROCEDURES

Materials—Lactacystin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dbcAMP, and all cell culture reagents were obtained from Sigma. Lipofectamine 2000, Zeocin, G418, ponasterone A, and mouse monoclonal anti-v5 were purchased from Invitrogen. Rabbit polyclonal anti-ubiquitin was from Dako, and mouse monoclonal anti-GFP was from Roche Applied Science. Goat anti-mouse IgG-Cy3 was purchased from Molecular Probes and horseradish peroxidase-conjugated anti-mouse and anti-rabbit IgG were from Amersham Biosciences. Expression Plasmids and Stable Cell Lines—The enhanced green fluorescence protein (EGFP) and tNhtt expression constructs, pINDtNhtt-EGFP-16Q, pIND-tNhtt-150Q, and the generation of the stable cell lines of these constructs have been described previously [12]. The construction of plasmids, pEGFP-N1-MJD(f)-20CAG and pEGFP-N1MJD(f)-130CAG, pEGFP-N1-MJD(t)-20CAG, and pEGFP-N1-MJD(t)80CAG were described elsewhere [13]. The full-length CHIP cDNA was isolated from the total RNA extracted from HeLa cells by reverse transcription-PCR. Construction of full-length and the U-box-deleted CHIP in pcDNA vector with v5 tag were made using PCR. The stable cell lines (HD 16Q and HD 150Q) were maintained in the

CHIP Promotes Degradation of Polyglutamine Protein

RESULTS

DISCUSSION