UBE2E ubiquitin-conjugating enzymes and ubiquitin isopeptidase Y regulate TDP-43 ubiquitinylation [Molecular Bases of Disease]

May 13th, 2014 by Hans, F., Fiesel, F. C., Strong, J. C., Jackel, S., Rasse, T. M., Geisler, S., Springer, W., Schulz, J. B., Voigt, A., Kahle, P. J.

TDP-43 characterizes insoluble protein aggregates in distinct subtypes of frontotemporal lobar degeneration (FTLD-TDP) and amyotrophic lateral sclerosis (ALS). TDP-43 mediates many RNA processing steps within distinct protein complexes. Here we identify novel TDP-43 protein interactors in a yeast 2-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 ubiquitinylated, we focused on the ubiquitin-conjugating enzyme UBE2E3 and the ubiquitin isopeptidase UBPY. When cells were treated with proteasome inhibitor, ubiquitinylated and insoluble TDP-43 species accumulated. All three UBE2E family members could enhance the ubiquitinylation of TDP-43, whereas catalytically inactive [C145S]UBE2E3 was much less efficient. Conversely, silencing of UBE2E3 reduced TDP-43 ubiquitinylation. We examined 15 of the 34 known pathogenic TDP-43 mutants and found that one was excessively ubiquitinylated. This strong [K263E]TDP-43 ubiquitinylation was further enhanced by proteasomal inhibition as well as UBE2E3 expression. Conversely, UBE2E3 silencing and expression of UBPY reduced [K263E]TDP-43 ubiquitinylation. Moreover, wild-type but not active site mutant UBPY reduced ubiquitinylation 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 ubiquitinylation, solubility, and neurodegeneration.