Tumor Necrosis Factor Receptor-associated Factor 6 (TRAF6) Associates with Huntingtin Protein and Promotes Its Atypical Ubiquitination to Enhance Aggregate Formation

Francesca Persichetti,Alisia Carnemolla,Marta Codrich,Claudio Santoro,Marta Biagioli,Stefano Gustincich,Silvia Zucchelli,Elena Agostoni,Federica Marcuzzi,Sandra Vilotti,Milena Pinto

doi:10.1074/jbc.m110.187591

Abstract

Huntington disease (HD) is a neurodegenerative disorder caused by an expansion of polyglutamines in the first exon of huntingtin (HTT), which confers aggregation-promoting properties to amino-terminal fragments of the protein (N-HTT). Mutant N-HTT aggregates are enriched for ubiquitin and contain ubiquitin E3 ligases, thus suggesting a role for ubiquitination in aggregate formation. Here, we report that tumor necrosis factor receptor-associated factor 6 (TRAF6) binds to WT and polyQ-expanded N-HTT in vitro as well as to endogenous full-length proteins in mouse and human brain in vivo. Endogenous TRAF6 is recruited to cellular inclusions formed by mutant N-HTT. Transient overexpression of TRAF6 promotes WT and mutant N-HTT atypical ubiquitination with Lys6, Lys27, and Lys29 linkage formation. Both interaction and ubiquitination seem to be independent from polyQ length. In cultured cells, TRAF6 enhances mutant N-HTT aggregate formation, whereas it has no effect on WT N-HTT protein localization. Mutant N-HTT inclusions are enriched for ubiquitin staining only when TRAF6 and Lys6, Lys27, and Lys29 ubiquitin mutants are expressed. Finally, we show that TRAF6 is up-regulated in post-mortem brains from HD patients where it is found in the insoluble fraction. These results suggest that TRAF6 atypical ubiquitination warrants investigation in HD pathogenesis.

Highlights

Huntington disease (HD)2 is a dominantly inherited neurodegenerative disorder caused by the expansion of a polymorphic CAG sequence in the first exon of the gene encoding for huntingtin (HTT) protein [1]
These results suggest that tumor necrosis factor receptor-associated factor 6 (TRAF6) atypical ubiquitination warrants investigation in HD pathogenesis
E3 Ubiquitin Ligase TRAF6 Interacts with WT and Mutant HTT and Associates with Polyglutamine Aggregates—To evaluate whether TRAF6 might have a role in HD, we studied its ability to bind huntingtin amino-terminal fragments fused to green fluorescent protein (N-HTTGFP), with either a physiological (Gln21), or short (Gln60) or long (Gln150) pathological polyQ stretch

Summary

EXPERIMENTAL PROCEDURES

Plasmids and Cells—N-HTT-GFP Gln and Gln150 constructs were described previously [27]. Immunoprecipitation and Western Blot—For co-immunoprecipitation experiments, cells were lysed in TRAF6 buffer (200 mM NaCl, 50 mM Tris, pH 7.5, 0.5% Nonidet P-40, 10% glycerol) supplemented with anti-protease mixture (Roche Applied Science) and 5 mM N-ethylmaleimide. Cell lysates were incubated with anti-FLAG-agarose beads (Sigma) or with anti-GFP antibody (Invitrogen). Parietal cortex from HD patients was used for co-immunoprecipitation of endogenous proteins in the human brain sample. Cells were lysed directly in 2ϫ SDS sample buffer and analyzed by Western blot. Fractionation in soluble and insoluble protein extracts was performed from the cortex as follows; soluble fractions were extracted in 150 mM sucrose, 15 mM Hepes, pH 7.9, 60 mM KCl, 15 mM NaCl, 5 mM EDTA, 1 mM EGTA, 1% Triton X-100), supplemented with protease inhibitors (Roche Applied Science). The soluble fraction was removed, and the insoluble fractions were extracted from pel-

55 IP: HTT IB

RESULTS

DISCUSSION