Fig 1: Tau proteolysis map and in vitro fluorescence protease assays. a A peptide library tiling across tau 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 tau. The amino acid sequence of tau 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 7, so the cleavage occurs between positions 7 and 8). A total of 285 cleavages were found. Autosomal-dominant coding mutations associated with frontotemporal dementia are noted in red above the tau 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 tau 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 tau peptides. A grey box denotes a mutation which was predicted to be "non-damaging." Mutations disrupting protease cleave 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 rate cleavage (-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 tau peptide cleavage, comparing WT and mutant peptides as labeled (n = 3 for all protease-substrate pairs). N1 and N2, N-terminal repeats; P1 and P2, proline-rich regions, R1-4, microtubule binding repeats 1–4; Æ, asparagine endopeptidase (AEP)