Irreversible shift of TDP-43 into the cytoplasm alters its protein network

TDP-43 is an important DNA/RNA-binding protein that is mainly localized in the cell nucleus. In neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), it is mislocalized and becomes a central component of cytoplasmic protein aggregates. Our aim was to investigate the interactome of wild-type TDP-43 and TDP-43 without nuclear localization signal (dNLS), which mimics a pathological condition in ALS. We generated inducible mammalian cell lines expressing TDP-43wt and TDP-TDP-43dNLS. Using the BioID method, we investigated the interactome of these proteins, isolated biotinylated proteins by pull-down assays, and identified them by mass spectrometry. The resulting TDP-43wt and TDP-43dNLS interactors were validated. TDP-43wt predominantly interacted with proteins associated with ribonucleoproteins, spliceosome complexes and paraspeckles, whereas TDP-43dNLS interactors were associated with cytoplasmic stress granules and P-bodies. We also validated selected interacting proteins, namely NONO, SFPQ, FUS, MAML1, PUM1 and ATXN2L. We identified MAML1 as a unique TDP-43wt interactor, while NONO, SFPQ and FUS were common interactors that were more abundant in the TDP-43wt fraction. ATXN2L and PUM1 were found to be unique interactors of the TDP-43 mutant. Our results suggest a possible disruption of regulatory functions related to transcription and paraspeckle activity, as well as a link to stress granules and P-bodies in ALS development. In addition, the newly identified interactors will help to understand the aggregation process associated with TDP-43.