Figure 4

AChE associates with amyloid fibrils via the PrP N-terminus and affect their physical-chemical characteristics. This association is inhibited by Hup8TH. a Co-aggregation of AChE with preformed PrP fibrils (Fib PrP) was followed by Coomassie blue staining after SDS-PAGE of 10 μM Fib PrP mixed with 5 μM AChE at 37°C for 30 min. b AChE increases ANS binding to preformed PrP fibrils. ANS fluorescence emission spectra of monomeric PrP protein (dotted line), untreated PrP fibrils (long dashed line), fibrils after AChE association (short dashed line) and AChE alone (solid line). Samples correspond to either supernatant or pelleted fractions obtained after centrifugation. Final protein concentrations: 10 μM PrP, 5 μM AChE. Incubation conditions: 37°C for 30 min. c Altered digestion profile of the PK-resistant core of PrP fibrils after AChE binding. Silver staining following electrophoretic separation of PrP fibrils (10.5 μM ) incubated or not with AChE (0.5 μM), before and after PK digestion (+PK). The annealing procedure involves brief heating of the sample at 80°C in the presence of detergent. d Kinetics of AChE-PrP fibril co-assembly. Light scattering intensity was used to follow the increase in the average molecular weight during successive additions (indicated by an arrowhead) of 25 nM AChE to 50 nM preformed PrP fibrils. The vertical dotted line indicates when AChE was first added (time zero). Saturation occurs at equimolar concentrations. The profile obtained after a single addition of 50 nM AChE to the fibrillar sample is also shown to emphasize the equivalent total amplitude change. e AChE interacts with PrP fibrils obtained from two N-terminal truncated PrP variants (∆100-120 and ∆23-99) and reaches saturation at half the molar ratio (0.5:1, AChE:PrP), compared to full-length PrP fibrils. Two successive additions (indicated by an arrowhead) of 25 nM AChE to the two truncated PrP fibrils (50 nM) were performed. f Hup8TH decreases the binding of AChE to PrP fibrils. AChE was pre-incubated at room temperature with Hup8TH at a molar ratio of 1:20 (AChE:Hup8TH) for 30 min. Then, 50 nM PrP fibrils obtained from full-length PrP were incubated with 50 nM pre-treated (grey line with asterisk) or non-inhibited AChE (grey line without asterisks). The same experiment was performed using the two truncated PrP fibrils and with 25 nM (according to the saturation values observed in e) of pre-inhibited (black lines with asterisks) or not (black lines without asterisks) AChE. Data were normalized between 0 and 1 to easily compare the effects of the AChEi on the fraction of aggregated AChE. Experiments were performed in 20 mM Mes buffer (pH 6.0) at 37°C; a.u., arbitrary units. Molecular weights (in kilodaltons) are indicated to the left of the blots.