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 demonstrated 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 (3B). 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 
The composition of the aggresome was partially solved by mass spectrometry 
. Song and colleagues showed that the higher proportion of aggresome-enriched proteins is related to molecular chaperones and ubiquitin-proteasome system components, involved in the elimination of misfolded and/or ubiquitinated proteins from cells 
. 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 
. Among them are proteins known to interact with misfolded proteins and play a role in protein aggregation 
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 proteins were found ubiquitinated in previous studies 
or annotated in the Ubiprot database 
). 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 
. 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 
. 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) 
a modifier protein that plays a pivotal role in autophagosome biogenesis 
. Interestingly we have some preliminary data showing that TRIM50 and LC3 colocalize in both normal and autophagy-induced conditions (Figure S4B
and Fusco, unpublished results). Moreover using the NH4
Cl autophagy inhibitor, we found that TRIM50 is partially degraded through the autophagy-lysosomal pathway (). 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 
. Since aggresome formation mainly takes place in the insoluble fraction 
we assessed whether TRIM50 has a role in the accumulation of polyubiquitinted 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 (), 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 identification of TRIM50 specific substrates, are needed to unequivocally 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 
. 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) 
. 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.