Additional file 1 of Mutations in α-synuclein, TDP-43 and tau prolong protein half-life through diminished degradation by lysosomal proteases
Creators: name: Sampognaro, Paul J.
name: Arya, Shruti
name: Knudsen, Giselle M.
name: Gunderson, Emma L.
name: Sandoval-Perez, Angelica
name: Hodul, Molly
name: Bowles, Katherine
name: Craik, Charles S.
name: Jacobson, Matthew P.
name: Kao, Aimee W.
Publisher: name: figshare
Year: 2023
Subjects: subject: Biophysics
subject: Biochemistry
subject: 29999 Physical Sciences not elsewhere classified
subject: FOS: Physical sciences
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Neuroscience
subject: Evolutionary Biology
subject: 69999 Biological Sciences not elsewhere classified
subject: Cancer

description: Additional file 1: Figure S1. Proteases that do not cleave α-syn, TDP-43, or tau are active against a fluorogenic casein substrate. Fluorogenic protease activity assays using casein at pH 4.5 was performed on all enzymes that do not cleave α-syn, TDP-43, or tau. The values obtained were normalized to timepoint t = 0 and plotted relative to activity. All enzymes, including both cysteine and serine proteases, show activity against the optimized fluorogenic peptides compared to the substrate only control. Figure S2. PROSPER analysis predicts multiple cathepsin cleavage sites with α-syn, TDP-43, and tau. Cathepsins Gand K, the lysosomal proteases available in the PROSPER database, demonstrate many sites of cleavage throughout the α-syn, TDP-43, and tau substrates. Yellow highlighted amino acids represent the P1 site of predicted CTSK cleavage. Light blue highlighted amino acids represent the P1 of predicted CTSG cleavage. Figure S3. Cathepsin activity assay Vmax tables of α-syn, TDP-43, and tau wild type versus mutant substrates. a, α-Syn, b, TDP-43, and c, tau. Vmax values are listed in nM/min. An arbitrary value of 2000 value was given to protease-enzyme substrate interactions that were too fast to be modeled. Figure S4. Doxycycline-induced expression of mutant α-syn, TDP-43, and tau in differentiated SHSY-5Y cells. a, α-Syn; b, TDP-43; and c, tau. Western blot images demonstrating induced expression of each FLAG-tagged protein by our plasmid constructs with 24 h of doxycycline treatment results in measurable protein whose half-life can be readily quantified over days. These results also demonstrate minimal leakiness of these constructs in the absence of doxycycline. Figure S5. Proteasomal inhibitor, MG132, upregulates markers of autophagy. a., Western blot images demonstrating that Lamp1, cathepsin D, LCBIIb, and TFEB are all upregulated after MG132 treatment at 24, 72, and 120 h, compared to the no treatment condition. b., Quantification of the results of A. Experiments were performed in triplicate. Figure S6. Full Western blots of differentiated SH-SY5Y and iNeuron protein half-life experiments. a. Western blot images demonstrating induced expression of each FLAG-tagged protein whose half-lives were quantified over 5 days. The green channel was used to measure each FLAG tagged protein. The red channel was used to measure GAPDH, which was used as the loading control and method by which protein quantification was normalized. b. Western blot images demonstrating iNeurons whose protein levels were quantified at day 0 and day 5 of MG132 treatment. The green channel was used to measure either α-syn, TDP-43, or tau expression. The red channel was used to measure GAPDH, which was used as the loading control and method by which protein quantification was normalized. Figure S7. Table S1. α-Syn, TDP-43, and tau peptide library. Amino acid sequences for all peptides within the library tested in the described MSP-MS experiments in Figs. 2, 3, 4 and 5. Table S2. Pre-activation steps, protease concentrations, tested pHs, and timepoints for in vitro and mass spectrometry experiments. Cathepsin A, C, H, and X required pre-activation for all experiments in Figs. 1, 2, 3, 4, 5 and Fig. S1. Protease concentration and mass spectrometry timepoints were chosen and optimized based on the known rate of cleavage for each protease. Table S3. WT and mutant α-syn, TDP-43, and tau sequences with high rank order prediction scores of cleavage for select cathepsins. Each mutation and companion wild-type sequences for a, α-Syn; c, TDP-43, and c, tau are represented alongside the rank order prediction scores of PROSPER predicted sites of cleavage for the available cathepsins B, K, L, S, D and E. Table S4. Fluorogenic peptide sequences for WT and mutant α-syn, TDP-43, and tau. A, α-Syn; B, TDP-43, and C, tau amino acid sequences tested in the described fluorogenic protease activity assays in Figs. 2, 3, and 4.