Pirc Katja, Škarabot Miha, Pogačnik Lea, Žerovnik Eva, Poklar-Ulrih Nataša
Acta Chim Slov. 2015;62(1):181-9. doi: 10.17344/acsi.2014.882.
Aggregation of the intrinsically disordered protein α-synuclein into ordered amyloid fibrils is implicated in the pathogenesis of Parkinson's disease. To unravel the role of Tyr residues in α-synuclein fibrillation, we prepared recombinant N-terminal (Y39A) and C-terminal (Y(125,133,136)A) mutants of α-synuclein and examined their fibrillation propensities by thioflavin T and 1-anilinonaphthalene-8-sulfonate (ANS) fluorescent probes, SDS-PAGE and atomic force microscopy. We demonstrate that in contrast to wild-type α-synuclein, both mutants show large, but comparable delays in the fibrillation process and exhibit enhanced hydrophobicity during fibril-like assembly. Both Tyr mutants form fibril-like structures after prolonged incubation periods, which are morphologically distinct from those of the wild-type protein. Our results suggest that the N-terminal and C-terminal Tyr residues of α-synuclein are important primarily for the initiation of the fibrillation process.
内在无序蛋白α-突触核蛋白聚集成有序的淀粉样纤维与帕金森病的发病机制有关。为了阐明酪氨酸（Tyr）残基在α-突触核蛋白纤维化中的作用，我们制备了α-突触核蛋白的重组N端（Y39A）和C端（Y(125,133,136)A）突变体，并通过硫黄素T和1-苯胺基萘-8-磺酸盐（ANS）荧光探针、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和原子力显微镜检查了它们的纤维化倾向。我们证明，与野生型α-突触核蛋白相比，这两种突变体在纤维化过程中都表现出较大但相当的延迟，并且在类纤维组装过程中表现出增强的疏水性。两个酪氨酸突变体在长时间孵育后形成类纤维结构，其形态与野生型蛋白不同。我们的结果表明，α-突触核蛋白的N端和C端酪氨酸残基主要对纤维化过程的起始很重要。
