Quintas A, Vaz D C, Cardoso I, Saraiva M J, Brito R M
Centro de Neurociências de Coimbra, Universidade de Coimbra, 3004-517 Coimbra, the Instituto Superior de Ciências da Saúde Sul, Quinta da Granja, 2825 Monte da Caparica, Portugal.
J Biol Chem. 2001 Jul 20;276(29):27207-13. doi: 10.1074/jbc.M101024200. Epub 2001 Apr 16.
Amyloid fibril formation and deposition is a common feature of a wide range of fatal diseases including spongiform encephalopathies, Alzheimer's disease, and familial amyloidotic polyneuropathies (FAP), among many others. In certain forms of FAP, the amyloid fibrils are mostly constituted by variants of transthyretin (TTR), a homotetrameric plasma protein. Recently, we showed that transthyretin in solution may undergo dissociation to a non-native monomer, even under close to physiological conditions of temperature, pH, ionic strength, and protein concentration. We also showed that this non-native monomer is a compact structure, does not behave as a molten globule, and may lead to the formation of partially unfolded monomeric species and high molecular mass soluble aggregates (Quintas, A., Saraiva, M. J. M., and Brito, R. M. M. (1999) J. Biol. Chem. 274, 32943-32949). Here, based on aging experiments of tetrameric TTR and chemically induced protein unfolding experiments of the non-native monomeric forms, we show that tetramer dissociation and partial unfolding of the monomer precedes amyloid fibril formation. We also show that TTR variants with the least thermodynamically stable non-native monomer produce the largest amount of partially unfolded monomeric species and soluble aggregates under conditions that are close to physiological. Additionally, the soluble aggregates formed by the amyloidogenic TTR variants showed morphological and thioflavin-T fluorescence properties characteristic of amyloid. These results allowed us to conclude that amyloid fibril formation by some TTR variants might be triggered by tetramer dissociation to a compact non-native monomer with low conformational stability, which originates partially unfolded monomeric species with a high tendency for ordered aggregation into amyloid fibrils. Thus, partial unfolding and conformational fluctuations of molecular species with marginal thermodynamic stability may play a crucial role on amyloid formation in vivo.
淀粉样纤维的形成和沉积是多种致命疾病的共同特征，包括海绵状脑病、阿尔茨海默病和家族性淀粉样多神经病（FAP）等众多疾病。在某些形式的FAP中，淀粉样纤维主要由转甲状腺素蛋白（TTR）的变体构成，TTR是一种同四聚体血浆蛋白。最近，我们发现，即使在接近生理温度、pH、离子强度和蛋白质浓度的条件下，溶液中的转甲状腺素蛋白也可能解离为非天然单体。我们还发现，这种非天然单体是一种紧密结构，不像熔球态那样，可能会导致部分未折叠单体物种和高分子质量可溶性聚集体的形成（昆塔斯，A.，萨拉伊瓦，M. J. M.，和布里托，R. M. M.（1999年）《生物化学杂志》274卷，32943 - 32949页）。在此，基于四聚体TTR的老化实验和非天然单体形式的化学诱导蛋白质去折叠实验，我们表明，四聚体解离和单体的部分去折叠先于淀粉样纤维的形成。我们还表明，具有热力学上最不稳定非天然单体的TTR变体在接近生理的条件下产生的部分未折叠单体物种和可溶性聚集体数量最多。此外，由淀粉样变TTR变体形成的可溶性聚集体表现出淀粉样的形态和硫黄素 - T荧光特性。这些结果使我们得出结论，某些TTR变体形成淀粉样纤维可能是由四聚体解离为具有低构象稳定性的紧密非天然单体引发的，这种单体产生具有高度有序聚合成淀粉样纤维倾向的部分未折叠单体物种。因此，具有边缘热力学稳定性的分子物种的部分去折叠和构象波动可能在体内淀粉样形成中起关键作用。
