Amplification of distinct α-synuclein fibril conformers through protein misfolding cyclic amplification
Amplification of distinct α-synuclein fibril conformers through protein misfolding cyclic amplification
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DOI：
10.1038/emm.2017.1
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发表时间：
2017-04-01
影响因子：
12.8
通讯作者：
Lee, Seung-Jae
中科院分区：
文献类型：
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作者：
Jung, Byung Chul;Lim, Yoon-Ju;Lee, Seung-Jae
淀粉样原纤维的形成与神经退行性疾病的发病机制有关。原纤化产生许多构象异构体。据推测，构象异构体可能具有特定的生物学特性，从而为朊病毒菌株提供了生物化学框架。然而，各种原纤维构象和它们的致病功能之间的精确关系还没有确定，因为有限的可及性，从组织样品的足够量的原纤维。α-突触核蛋白就是这样一种蛋白质，它与帕金森病有关。使用称为蛋白质错误折叠循环扩增的技术，最初开发用于扩增朊病毒，我们建立了一个程序，通过该程序可以扩增α-突触核蛋白原纤维。用微量的种子，我们成功地扩增了α-突触核蛋白原纤维。即使在多轮循环扩增后，种子的复制在构象方面也是忠实的。此外，两个转基因小鼠品系，每一个代表一个不同的突触核蛋白病被用来调查不同的构象，通过使用这种技术。来自这两种菌株的组织提取物的扩增的α-突触核蛋白原纤维导致产生具有不同蛋白酶K消化谱的两种不同的原纤维构象异构体。总之，我们的研究结果表明，微量的α-突触核蛋白原纤维的组织提取物中可以扩增其构象保守。这种方法在扩增患有突触核蛋白病的患者的脑和体液中的α-突触核蛋白原纤维中应该是有用的，并且可以用作帕金森病和其他突触核蛋白病的潜在诊断工具。
Amyloid fibril formation has been implicated in the pathogenesis of neurodegenerative diseases. Fibrillation generates numerous conformers. Presumably, the conformers may possess specific biological properties, thus providing a biochemical framework for strains of prions. However, the precise relationship between various fibril conformers and their pathogenic functions has not been determined because of limited accessibility to adequate amounts of fibrils from tissue samples. a-Synuclein is one such protein, and it has been implicated in Parkinson disease. Using a technique known as protein misfolding cyclic amplification, originally developed for amplifying prions, we established a procedure through which the amplification of a-synuclein fibrils is possible. With a trace amount of seeds, we succeeded in amplifying alpha-synuclein fibrils. The replication of the seeds was faithful in terms of conformation even after multiple rounds of cyclic amplification. Moreover, two transgenic mouse strains each representing a distinct synucleinopathy were used to investigate different conformers by using this technique. The amplified a-synuclein fibrils derived from the tissue extracts of these two strains led to the production of two different fibril conformers with distinct proteinase K digestion profiles. Together, our results demonstrated that a trace amount of a-synuclein fibrils in tissue extracts could be amplified with their conformations conserved. This procedure should be useful in amplifying a-synuclein fibrils from the brains and body fluids of patients afflicted with synucleinopathies and may serve as a potential diagnostic tool for Parkinson disease and other synucleinopathies.