Fig. 3: EV-generated α-syn fragments have reduced seeding capacity.

A Coomasie blue stain of the EV-cleaved α-syn. Brackets indicate MW bands that were gel excised for proteomic analysis. The identified cleavage sites are depicted above the schematic representation of the α-syn molecule. The length of the bar represents the frequency of this cleavage based on the MS/MS spectra. The higher the length, the more common. Thus, the E114 is the most common cleavage site. Other cleavage sites are found inside the NAC domain or at the C-terminus. Below the schematic structure of the α-syn molecule the Lys are marked, and the double-headed arrows represent the peptides that were identified in MS/MS. Cleavage at Lys is due to the semitryptic digest, while cleavage at other positions is due to proteases found in EVs. Cleavages found in only one MS/MS have not been included. B Seeding of monomeric α-syn is inhibited in the presence of hPFFs pre-treated with EVs. Monomeric human α-syn was incubated with hPFFs pre-treated with EVs (orange) and hPFFs pre-treated with EVs in the presence of protease inhibitors (gray). Aggregation kinetics of reactions were monitored over time through ThT fluorescence measurements. PBS (blue) or monomeric α-syn as seeds (yellow) were used as negative and positive controls, respectively. hPFFs pre-treated by EVs suppressed the aggregation of monomer α-syn, while in the presence of protease cocktails inhibitors, the aggregation was promoted. Data are expressed as mean ± standard deviation (SD) of 3 independent experiments. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.001.