Fig 2: Disease-associated mutations alter cathepsin cleavage of A\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\beta$$\end{document}β region. A Expanded A\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\beta$$\end{document}β region of the MSP-MS cleavage map from Fig. 2. Dotted lines represent known \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\beta$$\end{document}β, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\alpha$$\end{document}α, and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\gamma$$\end{document}γ cleavage sites. Protective (green) and deleterious (red) variants are plotted above the primary sequence. A\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\beta$$\end{document}β numbering is shown in black. Site E693 is highlighted with a grey bar and the 9 amino acid region used for peptide cleavage assays is highlighted in blue. B–G E693 variants alter cleavage by lysosomal proteases. Wild-type, Arctic (E693G), and Dutch (E693Q) fluorogenic peptides were incubated with various cathepsins. Cleavage activity was determined by quantifying fluorescence over time. H Maximum reaction velocity (Vmax) of the cleavage assays shown in (B–G). RFU/min are plotted on a logarithmic scale. I Differentiated wild-type and E693 variant SH-SY5Y cells were collected at 0 and 5 days following doxycycline induction of APP expression. J Band intensity from (I) was quantified and normalized to GAPDH on Western Blots. Error bars represent ± SEM. n = 8; * p < 0.05, ** < 0.01, *** < 0.001

Fig 3: APP is enriched in the endolysosomal compartment and is cleaved by lysosomal proteases in vitro. A Differentiated SH-SY5Y neurons were fixed and immunostained with antibodies against organelle markers (magenta) and APP (green). Lamp1 and Lamp2, EEA1, and ATP5A1 are enriched in lysosomes, early endosomes, and mitochondria, respectively. Scale bar = 10 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\mu$$\end{document}μm. B Colocalization analysis was performed as described in methods. Percent APP overlap with each organelle marker was quantified and compared using a One-Way ANOVA followed by Bonferroni post-hoc analysis. * p < 0.05, ** < 0.01, *** < 0.001. C Undifferentiated (“U”) and differentiated (“D”) SH-SY5Y cells were lysed and cellular contents were separated via density gradient centrifugation. Lysosome depleted (“Lyso depl.”) and lysosome enriched (“Lyso enrich.”) fractions were compared with whole cell samples on a Western Blot after staining with antibodies against APP, Lamp1, and GAPDH. D Cortex and cerebellum tissue was collected from LysoTag mice and neuronal lysosomes were collected via immunoprecipitation. Purified lysosomes (“Lyso IP”) were blotted alongside the non-immunoprecipitated flow through (“Flow Thr”) and whole cell samples. E Silver staining of sAPP cleaved by various lysosomal proteases and at multiple pH levels. Blots of enzymes that did not cleave sAPP are shown in Figure S4

Fig 4: APP augments Tau cleavage by CTSG. A Activity assay measuring cleavage of a fluorogenic APP peptide in the presence of increasing Trypsin concentrations. B–C Activity assays measuring the cleavage of a fluorogenic tau-peptide (B) and FITC-labelled tau (C) by CTSG over time in the presence of either sAPP or BSA. D Recombinant human tau-441 was cleaved in vitro by CTSG in the presence of either sAPP or BSA and analyzed via Western Blot. E Quantification of (D), measuring tau (55 kDa) as well as a prominent cleavage product (40 kDa). A two-way repeated measures ANOVA followed by Holm-Bonferroni correction post-hoc correction. Error bars represent ± SEM. n = 3; * p < 0.05, ** < 0.01, *** < 0.001

Fig 5: Lysosomal proteases exhibit unique substrate specificities that are pH-sensitive. A–H iceLogos generated for select enzymes based on APP MSP-MS data (additional iceLogos available in Figures S8–S9). I Stacked barplots representing the number of cleavage sites tallied at each pH J Graphical overview of the APP-770 protein and a map of cleavage patterns across APP at varying pH levels. Dotted lines occur every 100 amino acids. E1, E2 = E1 and E2 domains, respectively. KPI = Kunitz Protease Inhibitor domain. TM = transmembrane. Histograms were generated at each experimental pH value and protease combination tested using MSP-MS. Bar heights indicate the relative number of cleavage sites for each condition (ie. the area under each plot = 100%)