Figure 2

The formation of an aggregation-prone complex via specific interaction of AChE with PrP N-terminal domain is partially inhibited by Hup8TH. a Kinetics of AChE-PrP assembly. Increasing concentrations of AChE were added to 5 μM PrP. AChE:PrP molar ratios are indicated next to the trace. The extent of protein aggregation was recorded by light scattering intensity (also in panels b, f and g). The results for AChE in the absence of PrP are not shown because they overlap with the kinetic profile of PrP alone. b Probing the specificity of the interaction by strengthening the ionic character of the buffer. AChE was added to 20 μM PrP in the presence of 200 mM NaCl. The AChE:PrP molar ratios are indicated next to the trace. The asterisk shows the kinetic profiles of AChE (lower curve) and PrP (higher curve) alone. c Negative-stained transmission electron micrographs of AChE (upper left panel), PrP (upper right panel) and a mix of both proteins (lower panel). The molar ratio used was 0.5:1 (AChE:PrP). Scale bar = 250 nm. d The presence of both PrP and AChE in the insoluble fraction (pellet, P) after centrifugation of a mixture of 10 μM PrP with 5 μM AChE (incubated at 37°C for 30 min) confirms the formation of PrP-AChE aggregates. Gels were stained with Coomassie Blue. S, soluble fraction. e BS³ cross-linking assay demonstrates the formation of a heterodimer. Silver-stained gel showing PrP (10 μM), AChE (5 μM), and a mix of both proteins after incubation with 500 μM BS³. f Importance of the PrP N-terminus for the heterologous interaction. Left panel: schematic representation of the various PrP constructs. Right panel: kinetics of AChE assembly with truncated PrP variants. 50 nM AChE was added to 2 μM PrP variants (the full-length PrP profile is shown as grey line). The vertical dotted line indicates when AChE was added (time zero). g Hup8TH decreases AChE binding to PrP. AChE was pre-incubated at room temperature with increasing concentrations of Hup8TH for 30 min (AChE:Hup8TH molar ratios from 1:0.6 to 1:25). Then, 250 nM PrP was incubated with 50 nM pre-treated or not (grey line) AChE (AChE:Hup8TH molar ratios are indicated next to the trace). The asterisk highlights the kinetic profiles of AChE and PrP alone incubated with the highest Hup8TH concentration. h The effect of Hup8TH on AChE-PrP co-aggregation was monitored by Coomassie blue staining after SDS-PAGE. 10 μM PrP and 5 μM AChE were incubated or not at 37°C with Hup8TH (1:100 molar ratio, AChE:Hup8TH) for 30 min. Supernatant and pelleted fractions obtained after centrifugation were analyzed. 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.