The impact of the signal cascades of TNKS-PI31-proteasome on TDP-43-related proteinopathy

Ubiquitinated TDP-43 inclusion is the pathogenetic hallmark in ALS and FTLD. An excess amount of TDP-43 impairs the 26S proteasome degradation system. Under normal conditions, 26S proteasome activity is regulated by both PI31 and TNKS proteins. TNKS is involved in TDP-43 proteinopathy, with its activity correlated with cytosolic TDP-43 accumulation. However, the functional roles and regulatory mechanisms among PI31, TNKS, and TDP-43 under normal and pathological conditions remain to be clarified. Here we used an immunofluorescent (IF) assay to study the subcellular distribution and colocalization of PI31, TNKS, and TDP-43. We observed that TNKS overexpression forms cytosolic aggregates, while TDP-43 overexpression results in a diffuse nuclear distribution with partial localization in the cytoplasm, where it colocalizes with TNKS. Furthermore, to confirm their interactions, we performed immunoprecipitation (IP) and FRET assays to investigate the protein-protein interaction patterns between PI31/TNKS and TDP-43/TNKS in HEK293T, and primary hippocampus neurons. Our findings indicated that TNKS directly interacts with both TDP-43 and PI31. Interestingly, the FRETc values between YFP-hTNKS and CFP-hTDP-43 were higher than those of YFP-hTNKS /CFP-hPI31 pairs, suggesting that the binding affinity between YFP-hTNKS and CFP-hTDP-43 may be stronger than that between YFP-hTNKS and CFP-hPI31. Moreover, in our RIPA/UREA extraction in NSC-34 cells, we found that overexpression of either CFP-hTDP-43-FL or CFP-hTDP-43-A315T significantly increased insoluble endogenous TNKS and PI31 proteins. Similarly, overexpression of Flag-hTNKS also significantly enhanced insoluble endogenous PI31 and both endogenous TDP-43, CFP-hTDP-43-Fl, and CFP-hTDP-43-A315T. Here we provided a comprehensive picture of the functional roles and regulatory mechanisms among PI31, TNKS, and TDP-43 under normal and pathological conditions.