Distinguishing crystal-like amyloid fibrils and glass-like amorphous aggregates from their kinetics of formation
Distinguishing crystal-like amyloid fibrils and glass-like amorphous aggregates from their kinetics of formation
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DOI：
10.1073/pnas.1208228109
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发表时间：
2012-08
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影响因子：
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通讯作者：
Y. Yoshimura;Yuxi Lin;H. Yagi;Young-Ho Lee;H. Kitayama;K. Sakurai;M. So;H. Ogi;H. Naiki;Y. Goto
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文献类型：
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作者：
Y. Yoshimura;Yuxi Lin;H. Yagi;Young-Ho Lee;H. Kitayama;K. Sakurai;M. So;H. Ogi;H. Naiki;Y. Goto
淀粉样原纤维和无定形聚集体是与蛋白质错误折叠疾病相关的两种类型的异常聚集体。虽然它们在形态上不同，但这两种形式往往被不加区别地对待。β2-微球蛋白（β 2 m）是一种导致透析相关淀粉样变性的蛋白质，在pH 2.5时根据NaCl浓度形成淀粉样纤维或无定形聚集体。我们比较了它们形成的动力学，通过测量硫磺素T荧光，光散射和8-苯胺基-1-萘磺酸盐荧光来监测。硫磺素T荧光专门监测淀粉样蛋白纤维化，而光散射和8-苯胺基-1-萘磺酸盐荧光监测淀粉样蛋白纤维化和无定形聚集。淀粉样纤维在过饱和溶液中通过成核依赖机制形成，类似于结晶。原纤化的滞后期减少搅拌或超声辐照搅拌后，消失与预先形成的原纤接种。相反，玻璃状无定形聚集体快速形成，没有滞后期。搅拌和接种均不加速无定形聚集。因此，通过监测动力学，我们可以区分晶体样淀粉样纤维和玻璃样无定形聚集体。溶解度和过饱和度将是进一步理解蛋白质异常聚集的关键因素。
Amyloid fibrils and amorphous aggregates are two types of aberrant aggregates associated with protein misfolding diseases. Although they differ in morphology, the two forms are often treated indiscriminately. β2-microglobulin (β2m), a protein responsible for dialysis-related amyloidosis, forms amyloid fibrils or amorphous aggregates depending on the NaCl concentration at pH 2.5. We compared the kinetics of their formation, which was monitored by measuring thioflavin T fluorescence, light scattering, and 8-anilino-1-naphthalenesulfonate fluorescence. Thioflavin T fluorescence specifically monitors amyloid fibrillation, whereas light scattering and 8-anilino-1-naphthalenesulfonate fluorescence monitor both amyloid fibrillation and amorphous aggregation. The amyloid fibrils formed via a nucleation-dependent mechanism in a supersaturated solution, analogous to crystallization. The lag phase of fibrillation was reduced upon agitation with stirring or ultrasonic irradiation, and disappeared by seeding with preformed fibrils. In contrast, the glass-like amorphous aggregates formed rapidly without a lag phase. Neither agitation nor seeding accelerated the amorphous aggregation. Thus, by monitoring the kinetics, we can distinguish between crystal-like amyloid fibrils and glass-like amorphous aggregates. Solubility and supersaturation will be key factors for further understanding the aberrant aggregation of proteins.