Fig. 3

TDP-43 proteolysis map and in vitro fluorescence protease assays. a A peptide library tiling across TDP-43 was incubated with individual lysosomal proteases (left). At various times and pHs, the reaction was subjected to MSP-MS to detect proteolytic cleavage sites in TDP-43. The amino acid sequence of TDP-43 is in black letters at the top. Cleavage sites are indicated with the enzyme letter (e.g., B for CTSB) positioned at the P1 position (e.g. the B for CTSB is at amino acid position 17, so the cleavage occurs between positions 17 and 18). A total of 553 cleavages were found. Autosomal-dominant coding mutations associated with amyotrophic lateral sclerosis are noted in red above the TDP-43 sequence. Grey bars highlight amino acid mutations tested in in vitro fluorescent protease assays. b Pie chart demonstrating the number of cleavages sites within the TDP-43 sequence for each protease with the percentage of contributed cleavage sites in parentheses. c Table of maximal velocity (Vmax) ratios (mutant/WT), comparing protease cleavage of WT versus mutant TDP-43 peptides. A grey box denotes a mutation which was predicted to be "non-damaging." Mutations decreasing the Vmax of protease cleavage by 0–25% (1 point), 25–75% (2 points), and > 75% (3 points) are highlighted in light pink, dark pink, and dark red, respectively. Mutations augmenting the cleavage rate (-1 point) are highlighted in light green. Mutations with similar rate of cleavage compared to WT (0 points) are highlighted in yellow. Grey boxes denote no observed cleavage for either the WT or mutant peptide. Points were summed to derive a total “damage score”. d-i Representative curves of fluorescence generated from TDP-43 peptide cleavage, comparing WT and mutant peptides as labeled (n = 3 for all protease-substrate pairs). NLS, nuclear localization sequence; RRM1 and RRM2, RNA recognition motifs 1 and 2; Æ, asparagine endopeptidase (AEP)