Division of Bioanalytical Chemistry, Department of Chemistry and Pharmaceutical Sciences, Amsterdam Institute of Molecular and Life Sciences, Vrije Universiteit Amsterdam, De Boelelaan 1105, 1081 HV Amsterdam, The Netherlands.
Centre for Analytical Sciences Amsterdam, 1098 XH Amsterdam, The Netherlands.
ACS Chem Neurosci. 2024 Sep 18;15(18):3391-3400. doi: 10.1021/acschemneuro.4c00404. Epub 2024 Aug 30.
The aggregation of the proteins tau and amyloid-β is a salient feature of Alzheimer's disease, the most common form of neurodegenerative disorders. Upon aggregation, proteins transition from their soluble, monomeric, and functional state into insoluble, fibrillar deposits through a complex process involving a variety of intermediate species of different morphologies, including monomers, toxic oligomers, and insoluble fibrils. To control and direct peptide aggregation, a complete characterization of all species present and an understanding of the molecular processes along the aggregation pathway are essential. However, this is extremely challenging due to the transient nature of oligomers and the complexity of the reaction networks. Therefore, we have employed a combined approach that allows us to probe the structure and kinetics of oligomeric species, following them over time as they form fibrillar structures. Targeting the tau protein peptide segment Ac-PHF6-NH, which is crucial for the aggregation of the full protein, soft nano-electrospray ionization combined with ion mobility mass spectrometry has been employed to study the kinetics of heparin-induced intact oligomer formation. The oligomers are identified and characterized using high-resolution ion mobility mass spectrometry, demonstrating that the addition of heparin does not alter the structure of the oligomeric species. The kinetics of fibril formation is monitored through a Thioflavin T fluorescence assay. Global fitting of the kinetic data indicates that secondary nucleation plays a key role in the aggregation of the Ac-PHF6-NH tau segment, while the primary nucleation rate is greatly accelerated by heparin.
蛋白质 tau 和淀粉样蛋白-β的聚集是阿尔茨海默病（最常见的神经退行性疾病形式）的一个显著特征。聚集后，蛋白质通过涉及多种不同形态中间物的复杂过程，从可溶性、单体和功能性状态转变为不溶性、纤维状沉积物，这些中间物包括单体、毒性寡聚物和不溶性纤维。为了控制和指导肽聚集，必须对所有存在的物种类进行完整的表征，并了解沿聚集途径的分子过程。然而，由于寡聚物的瞬态性质和反应网络的复杂性，这极具挑战性。因此，我们采用了一种组合方法，使我们能够探测寡聚物的结构和动力学，并随着它们形成纤维状结构而随时间跟踪它们。针对对全长蛋白质聚集至关重要的 tau 蛋白肽段 Ac-PHF6-NH，采用软纳喷雾电离结合离子淌度质谱法来研究肝素诱导完整寡聚物形成的动力学。使用高分辨率离子淌度质谱法鉴定和表征寡聚物，表明肝素的添加不会改变寡聚物的结构。通过 Thioflavin T 荧光测定法监测纤维形成的动力学。对动力学数据的全局拟合表明，次级成核在 Ac-PHF6-NH tau 片段的聚集中起着关键作用，而肝素极大地加速了初级成核速率。
