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

Plasmids and Cells

N-HTT-GFP Gln²¹ and Gln¹⁵⁰ constructs were described previously (27). N-HTT-GFP Gln⁶⁰ was a serendipitous construct obtained during Gln¹⁵⁰ plasmid preparation. It was sequence-verified and controlled during each preparation. FLAG-TRAF6 (WT and DN) and HA-ubiquitin (WT and mutants) were described elsewhere (26).

HEK 293 cells (Sigma) were maintained in culture in DMEM (Invitrogen) with 10% FBS (Sigma). Transfections were performed with the standard calcium phosphate method or with Lipofectamine 2000 (Invitrogen), as required.

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). For co-immunoprecipitation of endogenous proteins anti-HTT mouse monoclonal (Chemicon, catalog nos. MAB 2166 and MAB 5490) and rabbit polyclonal HP1 (27) antibodies were used. Cortex was dissected from homozygous Hdh^Q111 knock-in mice (28) or control littermates, snap-frozen on liquid nitrogen, and stored at −80 °C. Parietal cortex from HD patients was used for co-immunoprecipitation of endogenous proteins in the human brain sample. Lysates were prepared homogenizing tissue in modified TRAF6 buffer (150 mm NaCl, 50 mm Tris, pH 7.5, 0.5% Nonidet P-40, 10% glycerol) supplemented with anti-protease mixture. Total protein content was measured, and an equivalent amount was used for co-immunoprecipitation. After washing, immunoprecipitated proteins were eluted with 2× SDS sample buffer, boiled, and analyzed by Western blot. The following antibodies were used: anti-FLAG (1:2000; Sigma), anti-GFP (1:1000; Invitrogen), anti-HA (1:1000; from hybridoma supernatant, kindly provided by Dr. Licio Collavin), anti-HTT (Chemicon, catalog no. MAB 2166), anti-TRAF6 (Santa Cruz Biotechnology, catalog no. sc-7221), and anti-β-actin (1:5000; Sigma). For detection, anti-mouse-HRP and anti-rabbit-HRP (Dako) or protein A-HRP (Upstate) in combination with ECL (GE Healthcare) were used. For total protein extracts, cells were lysed directly in 2× SDS sample buffer and analyzed by Western blot.

Cell-based Ubiquitination Assay

For cell-based ubiquitination assays, HEK cells were transfected with HA-ubiquitin, FLAG-TRAF6 (WT or DN), and N-HTT-GFP constructs. After transfection, cells were lysed with radioimmune precipitation assay buffer (150 mm NaCl, 50 mm Tris, pH 7.5, 1% Triton X-100, 1% deoxycholic acid, 0.1% SDS), and samples were briefly sonicated. After centrifugation, clear lysates were immunoprecipitated with anti-GFP or anti-FLAG antibodies. Immunocomplexes were analyzed by Western blot using anti-HA antibody to detect ubiquitin conjugates. For development, protein A-HRP was used.

Immunocytochemistry

Indirect immunofluorescence was carried out following standard methods (29). Anti-FLAG (1:1000; SIGMA), anti-ubiquitin (1:50; Dako) and anti-TRAF6 (1:100; catalog no. sc-8409, Santa Cruz Biotechnology) antibodies were used. For detection, Alexa Fluor 405 (blue) or 594 (red) (Invitrogen) labeled anti-mouse or anti-rabbit antibodies were used. All images were collected using a confocal microscope (Leica TCS SP2). The analysis of aggregates was performed on high resolution images using ImageJ software. At least 200 cells from two independent experiments were counted and scored for percentage of cells with aggregates. For the analysis of aggregate size, only cells (n = 200) with comparable levels of TRAF6 expression, as determined by fluorescence intensity normalized to cell area, were used. N-HTT cytoplasmic staining was quantified on the same cells on a randomly selected area. Background fluorescence was quantified from an area placed outside the cells and was subtracted to each signal.

Human Post-mortem Brain Samples

Control and HD post-mortem brain tissues used in this study were collected by the Harvard Brain Tissue Resource Center, McLean Hospital (Belmont, MA) and described previously (30). HD brains were assigned Vonsattel grade 3 pathology (31). Post-mortem intervals were from 12 to 32 h for controls and from 8 to 30 h for HD brains. Total proteins were extracted from parietal cortex in 10% SDS. 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). After centrifugation, the soluble fraction was removed, and the insoluble fractions were extracted from pellets in 10% SDS. For Western blot analysis, anti-TRAF6 polyclonal (Santa Cruz Biotechnology, catalog no. sc-7221) and monoclonal (Santa Cruz Biotechnology, catalog no. sc-8409) antibodies were used.

Quantitative Real-time PCR

Total RNA from cortex of pathologically confirmed HD and control subjects was isolated using the TRIzol reagent (Invitrogen). cDNA was obtained from 1 μg of purified RNA using the iSCRIPT^TM cDNA Synthesis Kit (Bio-Rad). Quantitative real-time PCR was performed using SYBR-Green PCR Master Mix (Applied Biosystem). Expression of TRAF6 was analyzed as already described (26).

Statistical Analysis

All experiments were repeated in duplicate or more. Confocal images were analyzed with ImageJ software. Western blot scans were quantified with Adobe Photoshop CS3. Data represent the mean with standard deviation. When necessary, each group was compared individually with reference to the control group using Student's t test (Microsoft Excel software).

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 (residues 1–171) fused to green fluorescent protein (N-HTT-GFP), with either a physiological (Gln²¹), or short (Gln⁶⁰) or long (Gln¹⁵⁰) pathological polyQ stretch. N-HTT-GFP constructs were transfected in HEK 293 cells with FLAG-TRAF6 or FLAG-empty vector, as control. Immunoprecipitation of transfected cell lysates revealed a specific binding between TRAF6 and N-HTT with no evident selectivity for polyQ length (Fig. 1a). Reverse co-immunoprecipitation of transfected proteins confirmed that TRAF6 physically associated with N-HTT with a physiological and pathological polyQ stretch (Fig. 1b).

We then performed co-immunoprecipitation experiments in HEK 293 cells on endogenous proteins proving TRAF6 interaction with full-length WT HTT in vitro (Fig. 1c). The cortex from control WT and Hdh^Q111 knock-in mice were also analyzed, showing that TRAF6 specifically binds full-length HTT in vivo with no preference for WT or expanded polyQ-containing protein (Fig. 1d). This interaction was confirmed in vivo in the parietal cortex from HD post-mortem brains (Fig. 1e).

We then analyzed TRAF6 localization in cells that express WT or mutant N-HTT. WT N-HTT was located both in the cytoplasm and the nucleus, with a diffused staining throughout the cell. TRAF6 was mainly present in the cytoplasm and partially in the nucleus, where it co-localized with WT N-HTT (Fig. 1, f and g). Similar data were obtained with overexpressed and endogenous TRAF6. Overexpression of WT N-HTT did not alter TRAF6 cellular localization (data not shown). When transfected in cells, mutant N-HTT-GFP (Gln⁶⁰ and Gln¹⁵⁰) developed fluorescent aggregates, as expected, located in the cytoplasm and concentrated in perinuclear regions. The presence of mutant N-HTT aggregates induced the recruitment of TRAF6 at the site of N-HTT inclusions, with a more prominent accumulation at the border (Fig. 1, f and g).

Altogether, these results demonstrate that TRAF6 binds huntingtin in vitro and in vivo in a polyQ length-independent manner. Furthermore, mutant protein can sequester TRAF6 into cellular aggregates.

TRAF6 Facilitates Atypical Polyubiquitination of WT and Mutant N-HTT

The interaction between TRAF6 and N-HTT and their co-localization in polyglutamine aggregates raised the possibility that N-HTT might be target of TRAF6 E3 ubiquitin ligase activity. To test this hypothesis, we performed ubiquitination assay in HEK 293 cells. Cells were transfected with HA-ubiquitin, N-HTT-GFP with 21, 60, or 150 polyQ segment and with or without FLAG-TRAF6. A TRAF6 mutant deleted of the N-terminal E3 ligase domain (DN) also was included as control. We found that overexpression of WT TRAF6, but not of DN mutant, facilitated the formation of high molecular weight polyubiquitinated forms of N-HTT (Fig. 2). TRAF6 activity was evident toward N-HTT with physiological length of polyQ stretch (Gln²¹) and for pathological mutants (Gln⁶⁰ and Gln¹⁵⁰). Therefore, TRAF6 ubiquitin-ligase activity did not seem to be polyQ-dependent, as already seen for its binding capability.

Because we have shown recently that TRAF6 can promote unconventional ubiquitin linkages on target proteins (26), we investigated the mode of polyubiquitination mediated by TRAF6 onto N-HTT. We thus took advantage of a set of ubiquitin mutants that we have generated with only one lysine available for chain formation (remaining lysine residues were substituted with arginine). An ubiquitin mutant with all lysines substituted with arginine was also included as negative control (Lys⁰). Cell-based ubiquitination assay was performed with all N-HTT constructs with or without TRAF6 and in combination with ubiquitin mutants. TRAF6 promoted a robust polyubiquitination of WT N-HTT in the presence of Lys⁶, Lys²⁷, and Lys²⁹ mutants. Background signals were observed with Lys⁰ mutant (Fig. 3a). Similar results were obtained with mutant N-HTT with short (Gln⁶⁰) or long (Gln¹⁵⁰) polyQ stretch (Fig. 3, b and c).

Therefore, we conclude that TRAF6 promotes an atypical polyubiquitination of WT and mutant N-HTT that is independent from polyQ length. Lys⁶, Lys²⁷, and Lys²⁹ are the preferred lysine residues for linkage formation.

TRAF6-mediated Ubiquitination Increases Mutant N-HTT Aggregates

When expressed in cells, Gln⁶⁰ and Gln¹⁵⁰ mutant, but not Gln²¹ N-HTT, generated aggregates, with an average size and number that correlate with polyQ length. We examined the influence of TRAF6-mediated ubiquitination on mutant N-HTT aggregation by transient transfection in HEK 293 cells. We included WT N-HTT as an internal reference in all our experiments. Only cells with comparable levels of TRAF6 expression (WT and DN), as determined by fluorescence intensity normalized to cell area, were used for analysis of aggregate number and size. The residual diffuse cytoplasmic staining was also quantified as additional indication of protein segregation into aggregates. TRAF6 overexpression had no influence on WT N-HTT cellular localization, with a diffuse cytoplasmic and nuclear staining as expected (Fig. 4a). Mutant N-HTT formed cytoplasmic and perinuclear aggregates, whose number and size correlated with polyQ length (Fig. 4a). Overexpression of TRAF6 increased the percentage of cells with aggregates and promoted the formation of inclusions with an average larger size (Fig. 4, b and c). This is likely due to a redistribution of the mutant protein to inclusions, as total protein levels were not affected by TRAF6 (supplemental Fig. S1) (compatible with a nondegradative role of atypical ubiquitination) and diffuse cytoplasmic staining decreased when aggregate number and size increased (Fig. 4d). The expression of TRAF6 DN mutant had no effect on percentage, size, and distribution of N-HTT aggregates, thus indicating that TRAF6 E3 ligase activity is required for its function on mutant N-HTT inclusion formation.

Because TRAF6 is recruited at the site of N-HTT aggregates and affects inclusion formation, we asked whether mutant protein might affect TRAF6 E3 ligase activity. To do so, we followed TRAF6 autoubiquitination by immunoprecipitation assay. We included WT N-HTT as an internal control. We found no differences in the levels of ubiquitinated TRAF6 when Gln²¹, Gln⁶⁰, or Gln¹⁵⁰ N-HTT was expressed (supplemental Fig. S2). Altogether, these data suggest a scenario in which TRA6-mediated ubiquitination may control N-HTT recruitment into aggregate structures without affecting its steady-state levels.

TRAF6-mediated Atypical Ubiquitination Is Involved in Mutant N-HTT Aggregates

To functionally link TRAF6-mediated atypical ubiquitination with mutant N-HTT aggregates, HEK 293 cells were transfected with HA-ubiquitin, FLAG-TRAF6, and N-HTT-GFP Gln¹⁵⁰. Aggregates were analyzed by double immunofluorescence coupled with GFP autofluorescence. First, we set the system using WT ubiquitin (Fig. 5a). Overexpression of ubiquitin per se enhanced N-HTT inclusion formation, as expected for the well established role of the UPS in mutant N-HTT aggregation. Nonetheless, under these conditions, ubiquitin staining was diffuse throughout the cell and did not accumulate at the site of N-HTT inclusions (Fig. 5a, upper panels). When TRAF6 was added to the system, all of the components coalesced into the aggregates, which appeared strongly stained with both ubiquitin and TRAF6 (Fig. 5a, lower panels). To prove that TRAF6 action on mutant N-HTT aggregates involved atypical ubiquitination, we carried out analogous experiments with ubiquitin mutants that were shown to be involved in N-HTT ubiquitination (Lys⁶, Lys²⁷, and Lys²⁹). As control, we used a Lys¹¹ mutant that mediates polychain formation but is not used by TRAF6. Lys⁶, Lys²⁷, and Lys²⁹ were able to recapitulate the phenotype observed with WT ubiquitin (Fig. 5b). On the contrary, the Lys¹¹ mutant did not accumulate into N-HTT inclusions. Together, these data provide evidence that TRAF6-mediated atypical activity is involved in controlling the composition of mutant N-HTT aggregates.

TRAF6 Is Up-regulated in Post-mortem Brains from HD Patients and Accumulates in Insoluble Protein Fraction

To obtain further evidence supporting the role of TRAF6 in HD pathogenesis, we assessed the expression of TRAF6 in human post-mortem brains from patients and age-matched healthy controls. First, TRAF6 mRNA expression was monitored by quantitative real-time PCR in RNA extracted from parietal cortex of controls and HD (Vonsattel grade 3 neuropathology) brains. TRAF6 mRNA levels were significantly up-regulated in HD compared with controls, with an average increase of 1.6-fold (Fig. 6a). We then tested whether TRAF6 up-regulation also was present at the protein level. Samples dissected from the parietal cortex of HD and control patients were used to prepare total protein extracts. Lysates were analyzed by Western blotting. To avoid artifacts due to unspecific signals, experiments were carried out using two different, commercially available anti-TRAF6 antibodies. Endogenous TRAF6 protein was slightly up-regulated in HD versus control samples (Fig. 6b). Quantification of TRAF6 signal demonstrated that the increase was statistically significant (Fig. 6c).

Because we found that mutant N-HTT promotes TRAF6 accumulation at the aggregates in cell culture system, TRAF6 distribution was studied in soluble and insoluble protein fractions prepared from human brain samples. Loading was normalized for total protein quantity in each of the two fractions. In HD and controls, TRAF6 was present mainly in the insoluble fraction. In this compartment, there was a statistically significant accumulation of TRAF6 in HD versus controls. Altogether, these data suggest an effect of mutant huntingtin on TRAF6 expression and solubility.