TRIM50 Promotes Aggresome Formation and Clearance Figure 4. TRIM50 colocalizes with p62 into aggresome. (A) SH-SY5Y cells were stained with anti-HDAC6 and with anti-p62 antibodies. The cells were treated with 25 mM MG132 for 6 h (d,e,f). (B) SH-SY5Y cells were stained with anti-TRIM50 and anti-p62 antibodies. The cells were treated with 25 mM MG132 for 6 h (d,e,f). (C) Lysates from FLAG-TRIM50#3 and FLAG#3 cell lines, treated with vehicle (–) or MG132 (+) were separated in RIPA detergent-soluble (S) and detergent insoluble (I) fractions and immunoblotting with anti-FLAG and anti-ubiquitin antibodies. (D) Lysates from MEF Trim50 cell lines with different genotype (+/+, 2/2), treated with vehicle (–) or MG132 (+) were separated in RIPA detergent-soluble (S) and detergent insoluble (I) fractions, analyzed by Western Blot and immunoblotting with anti-ubiquitin antibody. doi:10.1371/journal.pone.0040440.g004 proteins were found ubiquitinated in previous studies [29,30] or annotated in the Ubiprot database [31] (Table S2). Overall these data give additional evidences that TRIM50 bodies are aggresome precursors involved in the ubiquitination and aggregation process of misfolded proteins. We confirmed the interaction of TRIM50 with p62. p62 is a multifunctional adapter protein implicated in autophagy, cell signaling, receptor internalization, inflammation and protein turnover [22]. p62 is found in cytosolic protein aggregates that accumulate in various chronic, toxic, and degenerative diseases. It interacts with ubiquitinated proteins carrying them on the road to autophagy-mediated degradation [23,32]. The TRIM50-p62 interacting region involves amino acids 302–370, a region that includes the LC3-Interacting Region (LIR) domain involved in the binding to LC3 (microtubule-associated protein 1A/1B light chain 3) [23] a modifier protein that plays a pivotal role in autophago- some biogenesis [33]. Interestingly we have some preliminary data showing that TRIM50 and LC3 colocalize in both normal and autophagy-induced conditions (Figure S4B and Fusco, unpub- lished results). Moreover using the NH4Cl autophagy inhibitor, we found that TRIM50 is partially degraded through the autophagy- lysosomal pathway (Figure 6). However how this degradation occurs remains yet unclear; one possibility is that TRIM50 could directly associate with LC3, or that TRIM50 could be addressed together with p62 to autophagy machinery for its degradation. In that way the observed colocalization between TRIM50 and LC3 is intriguing and deserves more investigations. These findings suggest also that p62 may serve as a scaffold protein, via the interaction with TRIM50, whereby chains of polyubiquitin are transferred to target substrates for degradation. Nevertheless it is tempting to speculate that TRIM50 might be involved in autophagy processes as well. Increasing evidences indicate that autophagy-related proteins are sequestrated into the aggresome as a selective mechanism to regulate their degradation [24]. Since aggresome formation mainly takes place in the insoluble fraction [34,35] we assessed whether TRIM50 has a role in the accumulation of polyubiqui- tinted proteins. We observed that TRIM50 promotes the recruitment of polyubiquitinated proteins to aggresome and that the observed decrease of aggresome clearance was associated to the depletion of TRIM50 (Figure 5C), suggesting that these proteins are TRIM50 substrates. Overall the data reported in this study reveal a role for TRIM50 in aggresome formation and add further insights on its function by identifying and characterizing its first two protein partners. We speculate that, when the proteasome activity is impaired, TRIM50 ensures the sequestration of its targets to the aggresome via the association with HDAC6 and their subsequent likely removal by p62-mediated autophagy. Further studies, particularly the identi- fication of TRIM50 specific substrates, are needed to unequivo- cally assess the authenticity of this model. Accumulation of polyubiquitinated protein aggregates is a hallmark of several neurodegenerative disorders as well as of a number of other protein aggregation diseases affecting muscles, heart, liver and lung [36,37]. p62 has been identified as a component of inclusion bodies in several human diseases, such as neurodegenerative diseases (e.g., Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis) and in liver diseases (e.g., alcoholic hepatitis, hepatic steatosis, and hepatocellular carcinoma) [38]. It will hence be interesting to investigate whether TRIM50 is also a component of such bodies and it could even be responsible for targeting p62 to these sites. Materials and Methods Fusion Plasmids The pcDNA3-EGFP and pCDNA3-HA wild-type and mutants TRIM50 were described in [15]. Human b2-tubulin ORF was cloned into a pcDNA3 vector with FLAG as tag using a PCR based method with appropriate oligonucleotides followed by in- frame insertion into the vector. DsRED -LC3 and pENTR-EGFP, GST tagged p62 and GST-p62 mutants were a kind gift from Prof. T. Johansen (Institute of Medical Biology, University of Tromso, Norway), pcDNA3-Myc-p62 was a generous gift of Prof. Marie W. Wooten (Cellular and Molecular Biosciences Program, Auburn University, USA). pcDNA3-HA-HDAC6 mutants were a gift of Prof. Matthias (Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland). The plasmids used in this study are listed in Table S1. Cell Culture and Stable Cell Line Production HEK293, HeLa cells, SH-SY5Y (all from ATCC, Manassas, USA), and MEF were maintained in DMEM with Glutamax medium supplemented with 10% fetal bovine serum and 1% antibiotics (Invitrogen, Carlsbad, CA). Hdac6 MEF (mouse embryo fibroblast) cells were kindly provided by Prof. Joo-Yong Lee (Duke University, Durham, USA) [39]. Fugene 6 (Roche) was used for transfection according to the manufacturers’ instructions. HEK293 were transfected with pcDNA3-FLAG-TRIM50 or empty vector and selected for 2 weeks with 1 mg/ml G418 (Invitrogen, Carlsbad, CA) selective agent. The expressing colonies were expanded and then used for protein extract preparations following standard procedures. HEK293 cell line was used since the low level expression of endogenous TRIM50 protein. Hereafter the stable cell lines will be referred to as FLAG- TRIM50#3 and FLAG#3, respectively. Mouse Embryo Fibroblast Generation To identify loss-of-function mutation in Trim50, we screened the sequence of the first exon of this gene in the sperm DNA archives of F1 male progeny of ENU-treated (ethylnitrosourea) males and untreated females established by INGENIUM (http://www. ingenium-ag.com). We identified two missense (V55M and V60G) and one nonsense mutation. The latter mutation creates a premature stop codon by modifying TGC into a TGA amber codon. This C52X mutation in the middle of the RING domain abrogates the E3-ligase activity of Trim50, as the next in frame methionine residue is situated in the coiled-coil domain after both the RING and the B-box type 2 of the Trim50-encoded protein. We recovered the nonsense mutant mouse from the frozen archive using in vitro fertilization. Due to the generation method, the first PLoS ONE | www.plosone.org 8 July 2012 | Volume 7 | Issue 7 | e40440