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，通过均相结合形成细胞核（k_1）和原纤维自催化延伸（k_2）两步反应机制。本研究首先在HEPES缓冲液中观察到人降钙素原纤维形成的中间产物。我们进一步观察了HEPES溶液中中间体到原原纤维的转变过程。这一发现表明，在淀粉样蛋白纤维形成的过程中，通常会出现球形的中间产物，正如在a β-淀粉样蛋白肽中所观察到的那样。其次，研究了三种类型的原纤维抑制hCT的机制。首先制备了F16L和f19l - hct，并对其颤动动力学进行了研究。结果发现k_2在这些突变体中显著减少。其次，发现带电荷的氨基酸如Asp也能抑制hCT纤维的形成。在这种情况下，k_1显著降低，而k_2没有降低。第三，我们发现多酚化合物十字花科蛋白完全抑制了原纤维的形成。接下来，我们检查了胰高血糖素在脂质双层存在下的纤维形成，以了解活细胞中纤维的形成过程，因为胰高血糖素是29残基肽激素，已知在酸性条件下形成淀粉样纤维。与酸性溶液中淀粉样蛋白纤维的形成相比，k_2降低，k_1升高，说明脂质受到影响而形成淀粉样蛋白纤维。值得注意的是，在脂质双分子层存在时生长的胰高血糖素原纤维的结构与不存在脂质双分子层时生长的结构不同。即n端部分在原纤维形态中仍为α-螺旋，而在无脂质双分子层生长的原纤维中α-螺旋变为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.