Ban Tadato, Hoshino Masaru, Takahashi Satoshi, Hamada Daizo, Hasegawa Kazuhiro, Naiki Hironobu, Goto Yuji
Institute for Protein Research, Osaka University and CREST, Japan Science and Technology Agency, Yamadaoka 3-2, Suita, Osaka 565-0871, Japan.
J Mol Biol. 2004 Nov 26;344(3):757-67. doi: 10.1016/j.jmb.2004.09.078.
Amyloid fibril formation is a phenomenon common to many proteins and peptides, including amyloid beta (Abeta) peptide associated with Alzheimer's disease. To clarify the mechanism of fibril formation and to create inhibitors, real-time monitoring of fibril growth is essential. Here, seed-dependent amyloid fibril growth of Abeta(1-40) was visualized in real-time at the single fibril level using total internal reflection fluorescence microscopy (TIRFM) combined with the binding of thioflavin T, an amyloid-specific fluorescence dye. The clear image and remarkable length of the fibrils enabled an exact analysis of the rate of growth of individual fibrils, indicating that the fibril growth was a highly cooperative process extending the fibril ends at a constant rate. It has been known that Abeta amyloid formation is a stereospecific reaction and the stability is affected by l/d-amino acid replacement. Focusing on these aspects, we designed several analogues of Abeta(25-35), a cytotoxic fragment of Abeta(1-40), consisting of l and d-amino acid residues, and examined their inhibitory effects by TIRFM. Some chimeric Abeta(25-35) peptides inhibited the fibril growth of Abeta(25-35) strongly, although they could not inhibit the growth of Abeta(1-40). The results suggest that a more rational design of stereospecific inhibitors, combined with real-time monitoring of fibril growth, will be useful to invent a potent inhibitor preventing the amyloid fibril growth of Abeta(1-40) and other proteins.
淀粉样纤维形成是许多蛋白质和肽共有的现象，包括与阿尔茨海默病相关的淀粉样β（Aβ）肽。为了阐明纤维形成的机制并开发抑制剂，实时监测纤维生长至关重要。在此，使用全内反射荧光显微镜（TIRFM）结合淀粉样特异性荧光染料硫黄素T，在单纤维水平实时观察了Aβ（1-40）的种子依赖性淀粉样纤维生长。纤维清晰的图像和显著的长度使得能够精确分析单个纤维的生长速率，表明纤维生长是一个高度协同的过程，以恒定速率延伸纤维末端。已知Aβ淀粉样形成是一种立体特异性反应，其稳定性受l/d-氨基酸取代的影响。着眼于这些方面，我们设计了几种由l和d-氨基酸残基组成的Aβ（1-40）细胞毒性片段Aβ（25-35）的类似物，并通过TIRFM检测了它们的抑制作用。一些嵌合Aβ（25-35）肽强烈抑制Aβ（25-35）的纤维生长，尽管它们不能抑制Aβ（1-40）的生长。结果表明，结合纤维生长的实时监测，更合理地设计立体特异性抑制剂将有助于发明一种有效的抑制剂，防止Aβ（1-40）和其他蛋白质的淀粉样纤维生长。
