Secondary amyloidosis is a disease caused by a conformational change in serum amyloid A (SAA) protein, which is known to form amyloid fibrils with aggregates of abnormal β-sheets. To develop methods for preventing amyloidogenesis, it is necessary to understand the molecular basis of amyloid formation. Recently, we developed the mutation analysis for exploring amyloid-forming regions [1]. Based on the experimental results using a series of synthetic peptides, we were able to identify the essential interactions that are involved in amyloid formation. In this study, to reveal the molecular mechanism of amyloidogenesis of the SAA protein in detail, a series of synthetic peptides containing parts of the native sequence were analysed by a thioflavin T (ThT) -binding assay and by circular dichroism (CD) spectroscopy. The fluorescence enhancement of amyloid-bound ThT was shown in the peptides1-30, 21-50 and 31-60, including the regions of helix1 and helix 2, suggesting that amyloid fibrils had formed. The peptides forming amyloids resulted in a CD spectral pattern with positive bands at 215 −230 nm, which are specific to the cross-β structure. To narrow the search window, further screening was carried out by using truncated peptides and a series of diastereomeric point mutants with L- to D-amino acids substitution. The truncated peptides that included N-terminal 8 residues were found to be amyloidogenic fragments. In contrast, the diastereomeric mutations Arg1 and Phe4 showed no-amyloidogenic fragments. These results suggest that SAA1-8, especially Arg1 and Phe4 play essential roles in the molecular association.
To dissociate the SAA amyloid fibrils, a mid-infrared free-electron laser (FEL) was used to irradiate the amyloid-forming fragments. After being irradiated by FEL, the peptides showed lower intensity of fluorescence and reduction of the β-sheet. Thus, FEL irradiation was effective in dissociating SAA amyloid fibrils.
[1] Saiki, M., Hidaka, Y., Nara, M., Morii, H. (2012) Biochemistry, 51, 1566-1576.