The Regulation of TDP-43 Ubiquitinylation by UBE2E Ubiquitin-conjugating Enzymes and Ubiquitin Isopeptidase Y

Abstract

The TAR DNA binding protein of 43kDa (TDP-43) is the major component of insoluble protein aggregates in amyotrophic lateral sclerosis (ALS) and a subgroup of frontotemporal lobar degeneration (FTLD-TDP). Within these pathological aggregates TDP-43 is phosphorylated, ubiquitinylated and fragmented. The nucleic acid binding protein TDP-43 participates in mRNA splicing, stability, and transport as well as miRNA biogenesis. Therefore, this protein is part of distinct complexes whose functions are not fully understood. This study aimed to identify novel TDP-43 protein interactors, which may allow to get further insights into the functions of this protein. To this end, a yeast two-hybrid screen was performed utilizing as bait a C-terminal fragment (CTF) that is comprised of the RNA recognition motif 2 (RRM2) and the protein binding glycine-rich domain, and a human adult brain cDNA library. Ten positive clones with partial cDNAs were found, of which seven full-length cDNAs could be cloned. Their interactions with full-length TDP-43 and CTF were confirmed with coimmunoprecipitation and colocalization in human embryonic kidney (HEK293E) cells. TDP-43 is ubiquitinylated in pathological inclusions. Therefore, the roles of the class III E2 ubiquitin-conjugating enzyme UBE2E3 and the ubiquitin isopeptidase UBPY in ubiquitinylation of TDP-43 were further investigated. The inhibition of the proteasome in HEK293E cells resulted in the ubiquitinylation and a shift of TDP-43 into insoluble fractions. The three class III E2 enzymes UBE2E1, UBE2E2 and UBE2E3 can enhance the ubiquitinylation of TDP-43 upon overexpression, whereas the catalytically inactive UBE2E3 C145S failed to promote TDP-43 ubiquitinylation. Conversely, silencing of UBE2E3 reduced the amount of ubiquitinylated TDP-43. Additionally, the overexpression of UBPY reduced the ubiquitinylation of CTF and a nuclear import impaired TDP-43 mutant. This was dependent on the peptidase activity of UBPY, since two catalytically inactive mutants failed to reduce the ubiquitinylation of TDP-43. In this study the ubiquitinylation pattern of 15 out of 48 known pathogenic TDP-43 mutants was investigated. Only the TDP-43 K263E mutant was excessively ubiquitinylated. The ubiquitinylation of TDP-43 K263E was further enhanced upon proteasomal inhibition as well as UBE2E3 expression, but it was decreased by UBPY overexpression or UBE2E3 silencing. In Drosophila melanogaster, UBPY silencing in the eye enhanced a neurodegenerative TDP-43 phenotype and levels of insoluble higher molecular weight TDP-43 and ubiquitin were increased. In summary, UBE2E3 and UBPY regulate TDP-43 ubiquitinylation, solubility and possibly neurodegenerative effects. As such, UBPY might participate in decreasing pathological levels of aggregation-prone ubiquitinylated TDP-43.