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

人降钙素(Hct)是一种淀粉样蛋白形成肽，通过均相缔合成核(K_1)和纤维自身催化延长(K_2)两步反应机制被认为是淀粉样蛋白形成肽。在本研究中，首先在HEPES缓冲溶液中观察到了人降钙素原纤维形成的中间体。我们进一步观察了HEPES溶液中中间体向原纤维的转变过程。这一发现表明，在淀粉样原纤维形成过程中，通常会出现球形中间体，就像在Aβ-淀粉样多肽中观察到的那样。其次，研究了三种类型的纤维对Hct的抑制机理。首先，制备了F16L和F19L-hCTs，并研究了其原纤化动力学。结果表明，该突变体的k2显著降低。其次，发现荷电氨基酸如天冬氨酸也能抑制HCT纤维的形成。在这种情况下，k_1显著减小，而k_2没有减小。第三，我们发现多酚化合物十字花素完全抑制了原纤维的形成。接下来，我们研究了在脂肪双层存在的情况下高血糖素原纤维的形成，以及活细胞中的纤维形成过程，因为胰高血糖素是29个残基的多肽荷尔蒙，已知在酸性条件下形成淀粉样原纤维。在这种情况下，在酸性溶液中，与纤维形成相比，k_2降低，而k_1增加，表明脂质被影响形成淀粉样原纤维。值得注意的是，在有脂双分子层的情况下生长的胰高血糖素原纤维的结构与在没有脂双分子层的情况下不同。也就是说，在没有脂质双层的情况下，N-末端部分保持着α-螺旋的形式，而在没有脂双层的情况下，纤维中的α-螺旋转变为β-片状。

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.