UBE2E Ubiquitin-conjugating Enzymes and Ubiquitin Isopeptidase Y Regulate TDP-43 Protein Ubiquitination

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...

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Published in:	The Journal of biological chemistry Vol. 289; no. 27; pp. 19164 - 19179
Main Authors:	Hans, Friederike, Fiesel, Fabienne C., Strong, Jennifer C., Jäckel, Sandra, Rasse, Tobias M., Geisler, Sven, Springer, Wolfdieter, Schulz, Jörg B., Voigt, Aaron, Kahle, Philipp J.
Format:	Journal Article
Language:	English
Published:	United States Elsevier Inc 04-07-2014 American Society for Biochemistry and Molecular Biology
Subjects:	Adult Amyotrophic Lateral Sclerosis (ALS) (Lou Gehrig Disease) Animals Brain - metabolism DNA-Binding Proteins - metabolism Drosophila Drosophila melanogaster - metabolism Endopeptidases - deficiency Endopeptidases - metabolism Endosomal Sorting Complexes Required for Transport - deficiency Endosomal Sorting Complexes Required for Transport - metabolism Frontotemporal Dementia HEK293 Cells Humans Neurotoxins - metabolism Proteasome Protein Aggregation Protein Synthesis and Degradation Protein Transport Protein-Protein Interaction TDP-43 Two-Hybrid System Techniques Ubiquitin Thiolesterase - deficiency Ubiquitin Thiolesterase - metabolism Ubiquitin-conjugating Enzyme Ubiquitin-Conjugating Enzymes - metabolism Ubiquitin-specific Protease Ubiquitination Frontotemporal Dementia Ubiquitination Amyotrophic Lateral Sclerosis (ALS) (Lou Gehrig Disease) Protein Aggregation Drosophila Protein-Protein Interaction TDP-43 Ubiquitin-conjugating Enzyme Proteasome Ubiquitin-specific Protease
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Summary:	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 UBE2E3C145S 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-43K263E ubiquitination was further enhanced by proteasomal inhibition as well as UBE2E3 expression. Conversely, UBE2E3 silencing and expression of UBPY reduced TDP-43K263E 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. Background: Ubiquitin-modified TDP-43 protein aggregates characterize common neurodegenerative diseases. Results: UBE2E ubiquitin-conjugating enzymes and ubiquitin isopeptidase Y (UBPY) are functional interactors of TDP-43 in cell culture and fly models. Conclusion: Specific regulators of TDP-43 ubiquitination influence its aggregation and neurotoxic properties. Significance: UBE2E and UBPY enzymes may modulate the course of TDP-43 diseases.
Bibliography:	Present address: Dept. of Neuroscience, Mayo Clinic, Jacksonville, FL 32224. Present address: Dept. of Neuroscience, Mayo Clinic and Neurobiology of Disease Program, Mayo Graduate School, Jacksonville, FL 32224.
ISSN:	0021-9258 1083-351X
DOI:	10.1074/jbc.M114.561704