title: Coaggregation of RNA-binding proteins in a model of TDP-43 proteinopathy with selective RGG motif methylation and a role for RRM1 ubiquitination.
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.
authors: Department of Human Genetics, Emory University School of Medicine, Atlanta, Georgia, United States of America.
authors: Eric B
authors: EB
authors: Dammer
authors: Claudia
authors: C
authors: Fallini
authors: Yair M
authors: YM
authors: Gozal
authors: Duc M
authors: DM
authors: Duong
authors: Wilfried
authors: W
authors: Rossoll
authors: Ping
authors: P
authors: Xu
authors: James J
authors: JJ
authors: Lah
authors: Allan I
authors: AI
authors: Levey
authors: Junmin
authors: J
authors: Peng
authors: Gary J
authors: GJ
authors: Bassell
authors: Nicholas T
authors: NT
authors: Seyfried
mesh_terms: D020816:Amino Acid Motifs / Q000502:physiology*; D000818:Animals; D001120:Arginine / Q000737:chemistry; D015153:Blotting, Western; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005998:Glycine / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D008745:Methylation; D051379:Mice; D008810:Mice, Inbred C57BL; D009046:Motor Neurons / Q000166:cytology / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D055503:Protein Multimerization; D011499:Protein Processing, Post-Translational; D040901:Proteomics; D012333:RNA, Messenger / Q000235:genetics; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012262:Ribonucleoside Diphosphate Reductase; D057177:TDP-43 Proteinopathies / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D053719:Tandem Mass Spectrometry; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D004268:DNA-Binding Proteins; D010446:Peptide Fragments; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D025521:Tumor Suppressor Proteins; D001120:Arginine; C475286:RRM1 protein, human; D012262:Ribonucleoside Diphosphate Reductase; D005998:Glycine