Coaggregation of RNA-Binding Proteins in a Model of TDP-43 Proteinopathy with Selective RGG Motif Methylation and a Role for RRM1 Ubiquitination

• Type of Material: Article
• Authors: Eric B Dammer, Emory University; Claudia Fallini, Emory University; Yair M. Gozal, Emory University; Duc Duong, Emory University; Wilfried O Rossoll, Emory University; Ping Xu, Emory University; James J Lah, Emory University; Allan I Levey, Emory University; Junmin Peng, Emory University; Gary Bassell, Emory University; Nicholas Seyfried, Emory University
• Language: English
• Date: 2012-06-21
• Publisher: Public Library of Science
• Publication Version: Final Published Version
• Copyright Statement: © Dammer 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.
• License: Creative Commons Attribution 4.0 International
• Final Published Version (URL): http://dx.doi.org/10.1371%2Fjournal.pone.0038658
• Title of Journal or Parent Work: PLoS ONE
• Volume: 7
• Issue: 6
• Start Page: e38658
• End Page: e38658
• Grant/Funding Information: N.T.S was supported by a National Institute on Aging grant F32AG032848-02.; The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.; W.R. was supported by Muscular Dystrophy Association grant MDA173851.; This research project was supported in part by the Proteomics and Confocal Microscopy Core Facilities of the Emory Neuroscience NINDS Core Facilities grant, P30NS055077.; E.B.D. was supported by a National Institute on Aging grant F32AG038259 and National Institutes of Health T32 grant for translational neuroscience, NS-007480.
• Research Categories: Biology, Neuroscience; Health Sciences, Oncology; Biology, Cell

Abstract

TAR DNA-binding protein 43 (TDP-43) is a major component within ubiquitin-positive inclusions of a number of neurodegenerative diseases that increasingly are considered as TDP-43 proteinopathies. Identities of other inclusion proteins associated with TDP-43 aggregation remain poorly defined. In this study, we identify and quantitate 35 co-aggregating proteins in the detergent-resistant fraction of HEK-293 cells in which TDP-43 or a particularly aggregate prone variant, TDP-S6, were enriched following overexpression, using stable isotope-labeled (SILAC) internal standards and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). We also searched for differential post-translational modification (PTM) sites of ubiquitination. Four sites of ubiquitin conjugation to TDP-43 or TDP-S6 were confirmed by dialkylated GST-TDP-43 external reference peptides, occurring on or near RNA binding motif (RRM) 1. RRM-containing proteins co-enriched in cytoplasmic granular structures in HEK-293 cells and primary motor neurons with insoluble TDP-S6, including cytoplasmic stress granule associated proteins G3BP, PABPC1, and eIF4A1. Proteomic evidence for TDP-43 co-aggregation with paraspeckle markers RBM14, PSF and NonO was also validated by western blot and by immunocytochemistry in HEK-293 cells. An increase in peptides from methylated arginine-glycine-glycine (RGG) RNA-binding motifs of FUS/TLS and hnRNPs was found in the detergent-insoluble fraction of TDP-overexpressing cells. Finally, TDP-43 and TDP-S6 detergent-insoluble species were reduced by mutagenesis of the identified ubiquitination sites, even following oxidative or proteolytic stress. Together, these findings define some of the aggregation partners of TDP-43, and suggest that TDP-43 ubiquitination influences TDP-43 oligomerization.