The misfolding and aggregation of alpha-synuclein (aSyn) are thought to be central events in synucleinopathies. The physiological function of aSyn has been related to vesicle binding and trafficking, but the precise molecular mechanisms leading to aSyn pathogenicity are still obscure. In cell models, aSyn does not readily aggregate, even upon overexpression. Therefore, cellular models that enable the study of aSyn aggregation are essential tools for our understanding of the molecular mechanisms that govern such processes. Here, we investigated the structural features of SynT, an artificial variant of aSyn that has been widely used as a model of aggregation in mammalian cell systems, since it is more prone to aggregation than aSyn. Using Nuclear Magnetic Resonance (NMR) spectroscopy we performed a detailed structural characterization of SynT through a systematic comparison with normal, unmodified aSyn. Interestingly, we found that the conformations adopted by SynT resemble those described for the unmodified protein, demonstrating the usefulness of SynT as a model for aSyn aggregation. However, subtle differences were observed at the N-terminal region involving transient intra and/or intermolecular interactions that are known to regulate aSyn aggregation. Importantly, our results indicate that disturbances in the N-terminal region of SynT, and the consequent decrease in membrane binding of the modified protein, might contribute to the observed aggregation behavior of aSyn, and validate the use of SynT, one of the few models of aSyn aggregation in cultured cells.

中文翻译：

---

用于建模突触核蛋白病的α-突触核蛋白易聚集变体的分子表征。

α-突触核蛋白（aSyn）的错误折叠和聚集被认为是突触核蛋白病的中心事件。aSyn的生理功能已经与囊泡结合和运输有关，但导致aSyn致病性的确切分子机制仍然不清楚。在细胞模型中，即使过度表达，aSyn也不容易聚集。因此，能够研究aSyn聚集的细胞模型是我们了解控制这些过程的分子机制的重要工具。在这里，我们研究了SynT的结构特征，SynT是aSyn的人工变体，已被广泛用作哺乳动物细胞系统中的聚集模型，因为它比aSyn更易于聚集。使用核磁共振（NMR）光谱，我们通过与正常的未修饰aSyn进行系统比较，对SynT进行了详细的结构表征。有趣的是，我们发现SynT所采用的构象与未修饰蛋白所描述的构象相似，证明了SynT作为aSyn聚集模型的有用性。但是，在N端区域观察到细微的差异，涉及已知的调节aSyn聚集的瞬时内部和/或分子间相互作用。重要的是，我们的结果表明，SynT N端区域的紊乱以及随之而来的修饰蛋白膜结合力的下降可能有助于观察到的aSyn聚集行为，并验证了SynT的使用，这是少数模型之一培养细胞中aSyn聚集的变化。