TRIM50 Promotes Aggresome Formation and Clearance Figure 2. TRIM50 interacts with HDAC6 and p62. (A) The interaction between TRIM50 and HDAC6 was assayed in HEK293 FLAG-TRIM50#3 cell line, treated with MG132. The cell lysates were immunoprecipitated (IP) with anti-FLAG and Immunoblot (IB) with HDAC6 antibody. Asterisk indicate IgG aspecific band. (B) Schematic representation of TRIM50 deletion mutants used, with the minimal TRIM50 interaction region. (C) The interaction between TRIM50 and p62 was assayed in FLAG-TRIM50#3 transfected with Myc-p62. The cell lysates were immunoprecipitated with anti-FLAG (left) and with anti-Myc (right) antibodies, respectively. Immunoblot were done as indicated (asterisk indicates FLAG-TRIM50 coming from the first FLAG blotting). (D) Total lysates of HEK293 cells transfected both with TRIM50 deletion mutants and Myc-p62 were immunoprecipitated with anti-FLAG and immunoblotted with anti-Myc. doi:10.1371/journal.pone.0040440.g002 TRIM50 Promotes the Sequestration of Ubiquitinated Proteins into Aggresome and Drives the Accumulation of p62 and HDAC6 We investigated whether TRIM50 and p62 colocalize into the aggresome. First, by fluorescence microscopy we showed that p62 colocalizes with HDAC6 upon MG132 treatment (Figure 4A). Then we found a partial colocalization between endogenous p62 and TRIM50 in SH-SY5Y cells (Figure 4B, a–c), which was intensified in presence of MG132 (Figure 4B, d–f). Next we asked whether TRIM50 has any role in the recruitment and/or accumulation of polyubiquitinated proteins to the aggresome. MG132 treatment resulted in a prominent accumulation of higher-molecular-weight species constituted by polyubiquitinated proteins in the detergent insoluble fraction as showed by immunoblot with an anti-ubiquitin antibody (Figure 4C). These data were confirmed in Trim50 deficient mouse embryo fibroblasts (kindly provided by Prof. Reymond). Depletion of endogenous Trim50 resulted in a decrease of polyubiquitinated protein levels in MG132 treated cells (compare lane 4 to 8 in Figure 4D). We then examined whether the TRIM50 depletion influences the area, perimeter, and intensity of FK2-polyubiquitinated dots. MEF Trim502/2 cells showed a significant decrease of signal intensity, compared to the wild type and heterozygous counterpart, while no significant difference was observed for the area and size of the dots (Figure 5A). Notably, MEF Trim502/2 cells treated with MG132 showed a highly significant reduction of all three parameters compared to the wild type and heterozygous mouse cell lines, respectively (Figure 5B). Since the aggregates formation is a reversible process, to explore the effect of TRIM50 on the clearance of aggresome components, we analyzed the aggresome insolvency. MG132-pretreated MEF Trim50 cells were incubated in a free-drug media for 48 h, and the FK2-aggresome positive cells were counted. Immediately after the removal of MG132, we found a significant decrease of the number of FK2-positive aggregates in Trim502/2 cells compared to the Trim50+/+ (Figure 5C). More interestingly, 48 hours after the MG132 removal we observed a significantly higher number of FK2-positive aggregates in MEF Trim502/2 compared to MEF Trim50+/+ suggesting that Trim50 is required for the clearance of polyubiquitinated proteins included within aggresome (p = 0.03, Figure 5C). Thus our analysis suggests that TRIM50 plays an active role in the sequestration of polyubiquitinated proteins in the aggresome. Finally, we assessed whether TRIM50 overexpression has any effect on protein level of the endogenous HDAC6 and p62. p62 protein enrichment was observed in both soluble and insoluble fractions upon TRIM50 overexpression in the presence of MG132 (Figure S5A–B). Likewise we observed an increase of HDAC6 protein amount in both fractions, mainly upon MG132 treatment (Figure S5C). These results demonstrate that TRIM50 promotes the accumulation of both p62 and HDCA6, particularly into detergent insoluble aggregates. Inhibition of Lysosomal Activity Results in TRIM50 Accumulation and Increases the Number of TRIM50- positive Bodies To evaluate the role of the autophagy in the turnover of TRIM50, we measured the amount of TRIM50 protein level in cells treated or not with the lysosomal enzyme inhibitor ammonium chloride (NH4Cl) (Figure 6A–B). Compared with DMSO treated-cells, treatment with NH4Cl resulted in about 5 fold-increases in TRIM50 levels over time. Moreover we monitored the subcellular localization of TRIM50 cytoplasmic bodies after induction and inhibition of autophagic flux. In cells treated with NH4Cl, we observed a slight but clear increase in the number of intracellular TRIM50 positive bodies (Figure 6 C). Notably, FK2 positive proteins lose their colocalization with TRIM50 bodies after the inhibition of autophagic flux. These data suggested that TRIM50 itself and TRIM50-related bodies might be mainly degraded by autophagy-lysosomal pathway. Discussion In this study we showed that the E3 ubiquitin ligase TRIM50 forms highly dynamic and heterogeneous cytoplasmic bodies containing polyubiquitinated proteins. Inhibition of proteasome activity resulted in the coalescence of TRIM50 bodies into aggresome and in their colocalization with HDCA6 protein; this localization does not depend on the E3 ubiquitin ligase activity of TRIM50. Using fibroblast from Hdac6-deficient mice, we demon- strated that the TRIM50 aggresome localization is HDAC6- dependent. Importantly, in the presence of MG132, we observed that TRIM50 bodies change their shape in absence of HDAC6, becoming larger and lost the localization into the aggresome. Overall these evidences demonstrated that the TRIM50 inclusion bodies are aggresome precursors. Evidence that the TRIM50 localization is not merely artifact of overexpression comes from experiment in which the endogenous TRIM50 displayed a very similar localization to that of transfected protein (Figure 1B and S3B). Importantly this study demonstrated that TRIM50 is a novel component of and promotes the accumulation of ubiquitinated substrates to aggresome. Moreover we identified two novel TRIM50 partners, HDAC6 and p62, both involved in the clearance of polyubiquitinated and misfolded protein aggregates [2,3,4,24]. The composition of the aggresome was partially solved by mass spectrometry [25,26]. Song and colleagues showed that the higher proportion of aggresome-enriched proteins is related to molecular chaperones and ubiquitin-proteasome system components, in- volved in the elimination of misfolded and/or ubiquitinated proteins from cells [27]. Notably, a number of TRIM50 interactors that we have isolated in our proteomics approach, have been identified in a recent screening of proteins associated with MG132-induced aggresome in SH-SY5Y cells [26]. Among them are proteins known to interact with misfolded proteins and play a role in protein aggregation [28] including p62, chaperone proteins like Serpin H1, HSP90B1, PPIB, and 14-3-3 (eta and zeta) (Table S2). Moreover a number of the TRIM50-bound PLoS ONE | www.plosone.org 5 July 2012 | Volume 7 | Issue 7 | e40440