Oligomers of the β-amyloid peptide, Aβ, play a central role in the pathogenesis and progression of Alzheimer's disease. Trimers and higher-order oligomers composed of trimers are thought to be the most neurotoxic Aβ oligomers. To gain insights into the structure and assembly of Aβ oligomers, our laboratory has previously designed and synthesized macrocyclic peptides derived from Aβ17-23 and Aβ30-36 that fold to form β-hairpins and assemble to form trimers. In this study, we found that mutating Phe20 to cyclohexylalanine (Cha) in macrocyclic Aβ-derived peptides promotes crystallization of an Aβ-derived peptide containing the Aβ24-29 loop (peptide 3F20Cha) and permits elucidation of its structure and assembly by X-ray crystallography. X-ray crystallography shows that peptide 3F20Cha forms a hexamer. X-ray crystallography and SDS-PAGE further show that trimer 4F20Cha, a covalently stabilized trimer derived from peptide 3F20Cha, forms a dodecamer. Size exclusion chromatography shows that trimer 4F20Cha forms higher-order assemblies in solution. Trimer 4F20Cha exhibits cytotoxicity against the neuroblastoma cell line SH-SY5Y. These studies demonstrate the use of the F20Cha mutation to further stabilize oligomers of Aβ-derived peptides that contain more of the native sequence and thus better mimic the oligomers formed by full-length Aβ.

中文翻译：

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苯丙氨酸突变为环己基丙氨酸促进 Aβ 衍生的 β-发夹形成三角三聚体

β-淀粉样肽 (Aβ) 的寡聚物在阿尔茨海默病的发病机制和进展中发挥着核心作用。三聚体和由三聚体组成的高阶寡聚体被认为是最具神经毒性的Aβ寡聚体。为了深入了解Aβ寡聚体的结构和组装，我们的实验室之前设计并合成了源自Aβ17-23和Aβ30-36的大环肽，这些肽折叠形成β-发夹并组装形成三聚体。在这项研究中，我们发现，将大环 Aβ 衍生肽中的 Phe20 突变为环己基丙氨酸 (Cha) 可促进含有 Aβ24-29 环的 Aβ 衍生肽（肽 3F20Cha）的结晶，并允许通过 X 射线阐明其结构和组装晶体学。X 射线晶体学显示肽 3F20Cha 形成六聚体。X射线晶体学和SDS-PAGE进一步显示三聚体4F20Cha（衍生自肽3F20Cha的共价稳定三聚体）形成十二聚体。尺寸排阻色谱显示三聚体 4F20Cha 在溶液中形成高阶组装体。三聚体 4F20Cha 对神经母细胞瘤细胞系 SH-SY5Y 具有细胞毒性。这些研究证明，使用 F20Cha 突变可以进一步稳定 Aβ 衍生肽的寡聚物，这些肽含有更多的天然序列，从而更好地模拟全长 Aβ 形成的寡聚物。