Fig. 2

α-Syn proteolysis map and in vitro fluorescence protease assays. a A peptide library tiling across α-syn 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 α-syn. The amino acid sequence of α-syn 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 82 cleavages were found. Autosomal-dominant coding mutations associated with Parkinson’s Disease are noted in red above the α-syn sequence. Grey bars highlight amino acid mutations tested in in vitro fluorescent protease assays. b Pie chart demonstrating the number of cleavage sites within the α-syn 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 α-syn 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 α-syn peptide cleavage, comparing WT and mutant peptides as labeled (n = 3 for all protease-substrate pairs). NAC, non-amyloid component domain; Æ, asparagine endopeptidase (AEP)