Molecular mechanism of fibril formation in human calcitonin produced in the cell

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Human calcitonin(hCT)is known as an amyloid forming peptide by taking two steps reaction mechanism such as homogeneous association to form nucleus(k_1)and autocatalytic elongation of the fibrils (k_2). In this study, first, intermediate of the fibril formation of human calcitonin was observed in HEPES buffer solution. We have further observed the transition processes from intermediate to protofibril in the HEPES solution. This finding suggests that a spherical shape of intermediate is commonly occurred in the process of amyloid fibril formation as is observed in the Aβ-amyloid peptide. Second, three types of fibril inhibition mechanisms for hCT were investigated. First, F16L and F19L-hCTs were prepared and the fibrillation kinetics was examined. It turned out that the k_2 was significantly reduced in this mutants. Second, it was revealed that a charged amino acid such as Asp can also inhibit the fibril formation of hCT. In this case, k_1 was significantly reduced, while k_2 was not reduced. Third, we found that polyphenol compound crucmin completely inhibited the fibril formation. Next, we have examined the fibril formation of glucagon in the presence of lipid bilayers to underastand the fibril formation processes in the living cell, since lucagon is the 29-residue peptide hormone and is known to form amyloid fibril under the acidic condition. In this case k_2 was decreased, while k_1 was increased as compared with the fibril formation in the acidic solution, indicating that lipids were affected to form amyloid fibril. It is of interest to note that the structure of glucagons fibril grown in the presence of lipid bilayer is different from that in the absence of lipid bilayers. Namely, N-terminal part remains α-helix in the fibril form, while α-helix changed to β-sheet in the fibril grown in the absence of lipid bilayers.