title: Ubiquilin-2 (UBQLN2) binds with high affinity to the C-terminal region of TDP-43 and modulates TDP-43 levels in H4 cells: characterization of inhibition by nucleic acids and 4-aminoquinolines.
abstract: Recently, it was reported that mutations in the ubiquitin-like protein ubiquilin-2 (UBQLN2) are associated with X-linked amyotrophic lateral sclerosis (ALS), and that both wild-type and mutant UBQLN2 can co-localize with aggregates of C-terminal fragments of TAR DNA binding protein (TDP-43). Here, we describe a high affinity interaction between UBQLN2 and TDP-43 and demonstrate that overexpression of both UBQLN2 and TDP-43 reduces levels of both exogenous and endogenous TDP-43 in human H4 cells. UBQLN2 bound with high affinity to both full length TDP-43 and a C-terminal TDP-43 fragment (261-414 aa) with KD values of 6.2nM and 8.7nM, respectively. Both DNA oligonucleotides and 4-aminoquinolines, which bind to TDP-43, also inhibited UBQLN2 binding to TDP-43 with similar rank order affinities compared to inhibition of oligonucleotide binding to TDP-43. Inhibitor characterization experiments demonstrated that the DNA oligonucleotides noncompetitively inhibited UBQLN2 binding to TDP-43, which is consistent with UBQLN2 binding to the C-terminal region of TDP-43. Interestingly, the 4-aminoquinolines were competitive inhibitors of UBQLN2 binding to TDP-43, suggesting that these compounds also bind to the C-terminal region of TDP-43. In support of the biochemical data, co-immunoprecipitation experiments demonstrated that both TDP-43 and UBQLN2 interact in human neuroglioma H4 cells. Finally, overexpression of UBQLN2 in the presence of overexpressed full length TDP-43 or C-terminal TDP-43 (170-414) dramatically lowered levels of both full length TDP-43 and C-terminal TDP-43 fragments (CTFs). Consequently, these data suggest that UBQLN2 enhances the clearance of TDP-43 and TDP-43 CTFs and therefore may play a role in the development of TDP-43 associated neurotoxicity.
authors.affiliation: ALS Biopharma, LLC, Doylestown, PA 18902, USA. jcassel@alsbiopharma.com
authors.forename: Joel A
authors.initials: JA
authors.lastname: Cassel
authors.forename: Allen B
authors.initials: AB
authors.lastname: Reitz
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000634:Aminoquinolines / Q000494:pharmacology*; D052157:Aptamers, Nucleotide / Q000235:genetics / Q000378:metabolism; D000071183:Autophagy-Related Proteins; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods; D009154:Mutation / Q000187:drug effects; D009696:Nucleic Acids / Q000494:pharmacology*; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D054730:Protein Interaction Domains and Motifs; D014162:Transfection; D014452:Ubiquitins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D000634:Aminoquinolines; D052157:Aptamers, Nucleotide; D000071183:Autophagy-Related Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D009696:Nucleic Acids; D010446:Peptide Fragments; C405662:UBQLN2 protein, human; D014452:Ubiquitins; C001920:4-aminoquinoline
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