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.
Highlights
TAR DNA-binding protein 43 (TDP-43) is a major protein component in ubiquitin-positive, tau- and a-synuclein-negative inclusions of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) [1,2], which was initially identified due to its specific enrichment in the detergent-insoluble biochemical fraction of FTLD frontal cortex [1]
For this study, proteins potentially coaggregating with overexpressed TDP-43 or TDP-S6 were enriched and separated from potential contaminants, which were removed from the isolated aggregates by detergent-rich (RIPA) buffer extraction
This preceded quantification by unbiased mass spectrometry to identify and rank both covalent and non-covalent partners associated with the detergent insoluble fraction including detergent insoluble aggregates of TDP-43 or TDP-S6
Summary
TDP-43 is a major protein component in ubiquitin-positive, tau- and a-synuclein-negative inclusions of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) [1,2], which was initially identified due to its specific enrichment in the detergent-insoluble biochemical fraction of FTLD frontal cortex [1]. Physiological TDP-43 is a predominantly nuclear protein with the capacity to transiently shuttle to and from the cytoplasm in a manner dependent upon general transcription [3], pathological TDP-43 redistributes from the nucleus to the cytoplasm where it more often aggregates following phosphorylation, ubiquitination and proteolytic cleavage [1,2,4,5]. Cultured neurons and HEK-293 cells expressing full length TDP-43 consistently localize the protein almost exclusively to the nucleus [7]. Human TDP-S6 has 18 unique amino acids at its Cterminus and is 295 residues in total compared to the full length protein with 414 residues. The detergent-insoluble biochemical fraction for TDP-S6 expressing cells accumulated ubiquitin and SUMO2 or SUMO3 (SUMO2/3) conjugates at high-molecular weights, whereas the same fraction from TDP-43 overexpressing cells showed only an increase in SUMO2/3. Compared to TDPS6, TDP-43 was more robustly phosphorylated in western blots, consistent with phosphorylation on two C-terminal serine residues only present in TDP-43 [7,9]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
