UBE2E ubiquitin-conjugating enzymes and ubiquitin isopeptidase Y regulate TDP-43 protein ubiquitination

J Biol Chem. 2014 Jul 4;289(27):19164-79. doi: 10.1074/jbc.M114.561704. Epub 2014 May 13.

Abstract

Trans-activation element DNA-binding protein of 43 kDa (TDP-43) characterizes insoluble protein aggregates in distinct subtypes of frontotemporal lobar degeneration and amyotrophic lateral sclerosis. TDP-43 mediates many RNA processing steps within distinct protein complexes. Here we identify novel TDP-43 protein interactors found in a yeast two-hybrid screen using an adult human brain cDNA library. We confirmed the TDP-43 interaction of seven hits by co-immunoprecipitation and assessed their co-localization in HEK293E cells. As pathological TDP-43 is ubiquitinated, we focused on the ubiquitin-conjugating enzyme UBE2E3 and the ubiquitin isopeptidase Y (UBPY). When cells were treated with proteasome inhibitor, ubiquitinated and insoluble TDP-43 species accumulated. All three UBE2E family members could enhance the ubiquitination of TDP-43, whereas catalytically inactive UBE2E3(C145S) was much less efficient. Conversely, silencing of UBE2E3 reduced TDP-43 ubiquitination. We examined 15 of the 48 known disease-associated TDP-43 mutants and found that one was excessively ubiquitinated. This strong TDP-43(K263E) ubiquitination was further enhanced by proteasomal inhibition as well as UBE2E3 expression. Conversely, UBE2E3 silencing and expression of UBPY reduced TDP-43(K263E) ubiquitination. Moreover, wild-type but not active site mutant UBPY reduced ubiquitination of TDP-43 C-terminal fragments and of a nuclear import-impaired mutant. In Drosophila melanogaster, UBPY silencing enhanced neurodegenerative TDP-43 phenotypes and the accumulation of insoluble high molecular weight TDP-43 and ubiquitin species. Thus, UBE2E3 and UBPY participate in the regulation of TDP-43 ubiquitination, solubility, and neurodegeneration.

Keywords: Amyotrophic Lateral Sclerosis (ALS) (Lou Gehrig Disease); Drosophila; Frontotemporal Dementia; Proteasome; Protein Aggregation; Protein-Protein Interaction; TDP-43; Ubiquitin-conjugating Enzyme; Ubiquitin-specific Protease; Ubiquitination.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Animals
  • Brain / metabolism
  • DNA-Binding Proteins / metabolism*
  • Drosophila melanogaster / metabolism
  • Endopeptidases / deficiency
  • Endopeptidases / metabolism*
  • Endosomal Sorting Complexes Required for Transport / deficiency
  • Endosomal Sorting Complexes Required for Transport / metabolism*
  • HEK293 Cells
  • Humans
  • Neurotoxins / metabolism
  • Protein Transport
  • Two-Hybrid System Techniques
  • Ubiquitin Thiolesterase / deficiency
  • Ubiquitin Thiolesterase / metabolism*
  • Ubiquitin-Conjugating Enzymes / metabolism*
  • Ubiquitination*

Substances

  • DNA-Binding Proteins
  • Endosomal Sorting Complexes Required for Transport
  • Neurotoxins
  • Ubiquitin-Conjugating Enzymes
  • Endopeptidases
  • USP8 protein, human
  • Ubiquitin Thiolesterase