The E3-Ubiquitin Ligase TRIM50 Interacts with HDAC6 and p62, and Promotes the Sequestration and Clearance of Ubiquitinated Proteins into the Aggresome Carmela Fusco1, Lucia Micale1, Mikhail Egorov2, Maria Monti3, Ester Valentina D’Addetta1, Bartolomeo Augello1, Flora Cozzolino 3 , Alessia Calcagnı`1, Andrea Fontana4, Roman S. Polishchuk2, Gerard Didelot5, Alexandre Reymond5, Piero Pucci3, Giuseppe Merla1* 1 Medical Genetics Unit, IRCCS Casa Sollievo Della Sofferenza Hospital, San Giovanni Rotondo, Italy, 2 Telethon Institute of Genetics and Medicine, Naples, Italy, 3 CEINGE Advanced Biotechnology and Department of Organic Chemistry and Biochemistry, Federico II University, Napoli, Italy, 4 Unit of biostatistics, IRCCS Casa Sollievo Della Sofferenza Hospital, San Giovanni Rotondo, Italy, 5 Center for Integrative Genomics, University of Lausanne, Lausanne, Switzerland Abstract In this study we report that, in response to proteasome inhibition, the E3-Ubiquitin ligase TRIM50 localizes to and promotes the recruitment and aggregation of polyubiquitinated proteins to the aggresome. Using Hdac6-deficient mouse embryo fibroblasts (MEF) we show that this localization is mediated by the histone deacetylase 6, HDAC6. Whereas Trim50-deficient MEFs allow pinpointing that the TRIM50 ubiquitin-ligase regulates the clearance of polyubiquitinated proteins localized to the aggresome. Finally we demonstrate that TRIM50 colocalizes, interacts with and increases the level of p62, a multifunctional adaptor protein implicated in various cellular processes including the autophagy clearance of polyubiquitinated protein aggregates. We speculate that when the proteasome activity is impaired, TRIM50 fails to drive its substrates to the proteasome-mediated degradation, and promotes their storage in the aggresome for successive clearance. Citation: Fusco C, Micale L, Egorov M, Monti M, D’Addetta EV, et al. (2012) The E3-Ubiquitin Ligase TRIM50 Interacts with HDAC6 and p62, and Promotes the Sequestration and Clearance of Ubiquitinated Proteins into the Aggresome. PLoS ONE 7(7): e40440. doi:10.1371/journal.pone.0040440 Editor: Michael Sherman, Boston University Medical School, United States of America Received February 17, 2012; Accepted June 7, 2012; Published July 9, 2012 Copyright: 2012 Fusco et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was in part supported by grants from the MIUR FIRB Project Italian Human ProteomeNet RBRN07BMCT to PP, Je´roˆ me Lejeune Foundation, the Ricerca Corrente 2010 funding granted by the Italian Ministry of Health, the ‘‘561000’’ voluntary contributions, the Fondazione Banca del Monte di Foggia ‘‘Domenico Siniscalco Ceci’’, the Italian Telethon Foundation (Grant #GGP06122) and with the contribution of Ministero degli Affari Esteri, Direzione Generale per la Promozione e la Cooperazione Culturale (2009–2010) to GM; the European Commission anEUploidy Integrated Project (grant 037627), the Je´roˆ me Lejeune Foundation, the Swiss National Science Foundation to AR. We acknowledge the Telethon Electron Microscopy Core Facility (Telethon Grant #GFP08001) for the help with the electron microscopy. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: g.merla@operapadrepio.it Introduction The ubiquitin proteasome system (UPS) is a highly conserved pathway that removes non-functional, damaged, and/or misfolded proteins from the cell. However, when the capacity of the proteasome is impaired, misfolded proteins cannot be properly cleared and they accumulate into the aggresome [1,2], an inclusion body localized in the proximity of the microtubule- organizing centre (MTOC) [3,4]. Microtubule-associated histone deacetylase 6 (HDAC6) mediates this process [5]. Through its ubiquitin-binding BUZ finger domain, HDAC6 binds to and facilitates the transport of polyubiquitinated misfolded proteins along microtubules to aggresome [2]. Aggresome clearance is mediated by ubiquitin-binding proteins like p62/SQSTM1 and NBR1 [6]. These adaptor proteins through their ubiquitin-binding domain (UBA) decide the fate of protein degradation either through UPS or autophagy-lysosome pathway [6,7,8]. Ubiquitin ligases are terminal enzyme in the process of ubiquitination, which provides specificity to the pathway by recognizing the substrates. Experimental evidences suggest that E3-Ubiquitin ligases play an important role also in the execution of autophagy [9,10]. Therefore searching for new E3-Ubiquitin ligases involved in such processes are of interest. TRIM proteins are RING E3-Ubiquitin ligases defined by the presence of a tripartite motif consisting of a RING, one or two B-Box, and a Coiled-Coil domain involved in a variety of cellular processes, including regulation of cell cycle progression, differentiation, development, oncogenesis, and apoptosis [11,12,13]. TRIM50 is one of 28 hemizygous genes mapping to the region rearranged in Williams Beuren syndrome (WBS) [14,15], a genomic disorder characterized by mental retardation and multiple dysmorphic and metabolic features [16]. TRIM50 encodes an E3-Ubiquitin ligase that self-associates to form cytoplasmic bodies in the cell, like other TRIM proteins [13,15]. The nature and role of these bodies as well as the cellular function of TRIM50 is just beginning to emerge [15]. Here we report that TRIM50 cytoplasmic bodies are aggresome precursors. We show that during proteasome impairment TRIM50 promotes the recruitment and aggregation of poly- ubiquitinated proteins to the aggresome, and participates to aggresome clearance. In addition we identified two novel TRIM50 protein interactors, HDAC6 and p62, and show that PLoS ONE | www.plosone.org 1 July 2012 | Volume 7 | Issue 7 | e40440