Distinct Annular Oligomers Captured along the Assembly and Disassembly Pathways of Transthyretin Amyloid Protofibrils

BackgroundDefects in protein folding may lead to severe degenerative diseases characterized by the appearance of amyloid fibril deposits. Cytotoxicity in amyloidoses has been linked to poration of the cell membrane that may involve interactions with amyloid intermediates of annular shape. Although annular oligomers have been detected in many amyloidogenic systems, their universality, function and molecular mechanisms of appearance are debated. Methodology/Principal FindingsWe investigated with high-resolution in situ atomic force microscopy the assembly and disassembly of transthyretin (TTR) amyloid protofibrils formed of the native protein by pH shift. Annular oligomers were the first morphologically distinct intermediates observed in the TTR aggregation pathway. Morphological analysis suggests that they can assemble into a double-stack of octameric rings with a 16±2 nm diameter, and displaying the tendency to form linear structures. According to light scattering data coupled to AFM imaging, annular oligomers appeared to undergo a collapse type of structural transition into spheroid oligomers containing 8–16 monomers. Disassembly of TTR amyloid protofibrils also resulted in the rapid appearance of annular oligomers but with a morphology quite distinct from that observed in the assembly pathway. Conclusions/SignificanceOur observations indicate that annular oligomers are key dynamic intermediates not only in the assembly but also in the disassembly of TTR protofibrils. The balance between annular and more compact forms of aggregation could be relevant for cytotoxicity in amyloidogenic disorders.

研究背景：蛋白质折叠缺陷可引发以淀粉样纤维（amyloid fibril）沉积为特征的严重退行性疾病。淀粉样变性（amyloidoses）的细胞毒性与细胞膜穿孔相关，而该过程可能涉及与环状淀粉样中间体的相互作用。尽管已在多种淀粉样蛋白生成系统（amyloidogenic systems）中检测到环状寡聚体，但其普遍性、功能及形成的分子机制仍存在广泛争议。

研究方法与主要结果：我们采用高分辨率原位原子力显微镜（atomic force microscopy，AFM），研究了通过pH诱导天然蛋白转甲状腺素蛋白（transthyretin，TTR）构象改变所形成的淀粉样原纤维（amyloid protofibrils）的组装与解聚过程。环状寡聚体是TTR聚集通路中首个具有明确形态辨识度的中间体。形态学分析显示，此类寡聚体可组装成直径为16±2 nm的八聚环双堆叠结构，并倾向于形成线性聚集体。结合光散射数据与原子力显微镜成像结果，环状寡聚体似乎会经历坍塌型结构转变，形成包含8~16个单体的球状寡聚体。对TTR淀粉样原纤维的解聚实验同样快速产生了环状寡聚体，但其形态与组装通路中观察到的环状寡聚体存在显著差异。

结论与意义：本研究结果表明，环状寡聚体不仅是TTR原纤维组装过程中的关键动态中间体，同时也是其解聚过程中的关键动态中间体。环状聚集形式与更紧凑的聚集形式之间的平衡，或与淀粉样蛋白生成障碍性疾病（amyloidogenic disorders）的细胞毒性密切相关。