RESUMO
The cleavage of the amyloid precursor protein by ß- and γ-secretases is a key event in Alzheimer's disease. A fusion protein was constructed to investigate the cleavage rate and aggregation kinetics of amyloid-beta (1-40) (Aß(1-40)) peptides. The peptide was expressed with a Small Ubiquitin-Like Modifier (SUMO) on the N-terminus and cleaved by a SUMO protease Ulp1. The time course of the cleavage reaction was monitored by SDS-PAGE gel with 100:1 or 1000:1 SUMO-Aß(1-40) to Ulp1 molar ratio and in the presence of brain total lipid extract unilamellar vesicles. Similarly, the aggregation of Aß(1-40) peptides upon cleavage was monitored by thioflavin T fluorescence assays and by circular dichroism. The cleavage reaction was modulated by the concentration of Ulp1, with fast release of Aß(1-40) peptides producing shorter lag time before fibril formation, but with similar elongation rate. The presence of lipids significantly reduced the cleavage completion at 1000:1, but reduced the lag time before fibril formation, while at 100:1 similar cleavage and aggregation kinetics were observed compared to the lipid-free condition. Overall, the results showed that the fusion protein SUMO-Aß(1-40) is a means to study the cleavage and aggregation of amyloid peptides and that the presence of lipids and the fast release rate accelerated the aggregation of Aß(1-40) peptides.