Coaggregation of RNA-binding proteins in a model of TDP-43 proteinopathy with selective RGG motif methylation and a role for RRM1 ubiquitination

Eric B. Dammer, Claudia Fallini, Yair M. Gozal, Duc M. Duong, Wilfried Rossol, Ping Xu, James J. Lah, Allan I. Levey, Junmin Peng, Gary J. Bassell, Nicholas T. Seyfried

Research output: Contribution to journalArticle

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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.

Original languageEnglish (US)
Article numbere38658
JournalPLoS One
Volume7
Issue number6
DOIs
StatePublished - Jun 21 2012
Externally publishedYes

Fingerprint

RNA-binding proteins
Methylation
DNA-binding proteins
RNA-Binding Proteins
Ubiquitination
DNA-Binding Proteins
methylation
S 6
HEK293 Cells
detergents
Detergents
Agglomeration
ubiquitin
Ubiquitin
glycine (amino acid)
Poly(A)-Binding Protein I
Glycine
Proteins
ubiquitination
proteins

ASJC Scopus subject areas

  • Medicine(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)

Cite this

Coaggregation of RNA-binding proteins in a model of TDP-43 proteinopathy with selective RGG motif methylation and a role for RRM1 ubiquitination. / Dammer, Eric B.; Fallini, Claudia; Gozal, Yair M.; Duong, Duc M.; Rossol, Wilfried; Xu, Ping; Lah, James J.; Levey, Allan I.; Peng, Junmin; Bassell, Gary J.; Seyfried, Nicholas T.

In: PLoS One, Vol. 7, No. 6, e38658, 21.06.2012.

Research output: Contribution to journalArticle

Dammer, EB, Fallini, C, Gozal, YM, Duong, DM, Rossol, W, Xu, P, Lah, JJ, Levey, AI, Peng, J, Bassell, GJ & Seyfried, NT 2012, 'Coaggregation of RNA-binding proteins in a model of TDP-43 proteinopathy with selective RGG motif methylation and a role for RRM1 ubiquitination', PLoS One, vol. 7, no. 6, e38658. https://doi.org/10.1371/journal.pone.0038658
Dammer, Eric B. ; Fallini, Claudia ; Gozal, Yair M. ; Duong, Duc M. ; Rossol, Wilfried ; Xu, Ping ; Lah, James J. ; Levey, Allan I. ; Peng, Junmin ; Bassell, Gary J. ; Seyfried, Nicholas T. / Coaggregation of RNA-binding proteins in a model of TDP-43 proteinopathy with selective RGG motif methylation and a role for RRM1 ubiquitination. In: PLoS One. 2012 ; Vol. 7, No. 6.
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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.",
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AU - Duong, Duc M.

AU - Rossol, Wilfried

AU - Xu, Ping

AU - Lah, James J.

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