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 ubiqutin-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 6.2 nM and 8.7 nM, 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-immunoprecipation 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.

A High-Content Screen Identifies Novel Compounds That Inhibit Stress-Induced TDP-43 Cellular Aggregation and Associated Cytotoxicity

“…Dysfunction of two cellular catabolic processes, ubiquitination and autophagy, have been explored as possible mediators of TDP-43 aggregation. Ubiquilin-2, a component of the ubiquitin pathway, was recently shown to bind TDP-43 directly [23]. Disrupting this binding elicited a TDP-43 aggregation response.…”

Mutant UBQLN2 promotes toxicity by modulating intrinsic self-assembly

“…This phenomenon is observed for many RNA-binding proteins underlying multisystem proteinopathies and ALS/FTD (14). Several proteins in this group have been linked to UBQLN2: hnRNPA1 (15) and TDP43 (16) have been reported to interact with UBQLN2, and ubiquilins form a complex with VCP, an AAA+ ATPase that assists in the clearance of RNA granules (12,15,17). Together, these observations raise the possibility that UBQLN2 helps regulate RNA granule dynamics.…”

Structural Insights Into TDP-43 and Effects of Post-translational Modifications

“…The Aggregation-Prone C-Terminal Domain (CTD, Amino Acids 260-414) TDP-43 CTD is the site of about 50 disease-linked mutations (Mackenzie and Rademakers, 2008) as well as most of the phosphorylation sites (Figure 1), and has thus been examined extensively, but due to its disordered nature, all structural studies of the CTD have been of fragments. The TDP-43 CTD has been shown to be required for TDP-43 splicing activity (Ayala et al, 2005;Freibaum et al, 2010;Conicella et al, 2016), including autoregulation (Ayala et al, 2011), and is the site of interaction with several protein partners such as UBQLN2 (Cassel and Reitz, 2013), FMRP (Majumder et al, 2016) and hnRNP (Buratti et al, 2005;D'Ambrogio et al, 2009).…”