Figure 1. TDP-43 is subject to lysine acetylation within the RNA-binding domain.

a) QBI-293 cells were transfected with TDP-43 in the presence or absence of the acetyltransferase CBP and acetylated TDP-43 was evaluated by immunoprecipitation (anti-TDP-43 clone 171) followed by immunoblotting using anti-acetylated lysine antibody. b) Cells transfected with wild-type TDP-43 were pulse-labeled with [³H]-acetate for 2 hr followed by immunoprecipitation and analysis by SDS-PAGE and autoradiography for 2 weeks. Total cellular lysates representing 5% total input were immunoblotted using anti-TDP-43 and GAPDH antibodies. c) TDP-43 acetylation assay was performed similar to (a) above using either wild-type TDP-43 or the cytoplasmic mutant TDP-43-ΔNLS in the presence of the acetyltransferase CBP. Samples were analyzed by immunoprecipitation (top panel) and direct immunoblotting (bottom panels) using acetylated lysine and total TDP-43 antibodies. d) Double-labeling immunofluorescence was performed on cells transfected with CBP and TDP-43-ΔNLS using anti-HA and anti-myc antibodies, respectively. The merge indicates co-localization of CBP with cytoplasmic localized TDP-43. The scale bar represents 25 µm. e-f) Cells were transfected with vector alone or TDP-43-ΔNLS in the absence or presence of CBP and total TDP-43 was immunoprecipitated, separated by SDS-PAGE followed by gel excision, and analyzed by mass spectrometry, which identified the K145-acetylated peptide shown in f. g) CBP-mediated acetylation of two lysine residues clustered within the RNA-binding regions with significant ion scores and p-values are shown. h) Crystal structure analysis of RNA-binding domains RRM1 and RRM2 bound to DNA (Protein Data Bank entries 3D2W and 4IUF) illustrates Lys-145 and Lys-192 (shown in red) adjacent to critical Phe residues (shown in green) in the context of bound DNA (shown in cyan). i) Cytoplasmic TDP-43-ΔNLS or a comparable construct containing K→R mutations at Lys-145 and Lys-192 (TDP-43-ΔNLS-2KR) were analyzed by immunoblotting in the presence of CBP and HDAC inhibitors (nicotinamide/TSA) to promote full TDP-43 acetylation. The asterisk (*) indicates acetylated cross-reactive bands that are unaltered by TDP-43.