Maity Sibaprasad, Lyubchenko Yuri L
Department of Pharmaceutical Sciences, University of Nebraska Medical Center, 986025 Nebraska Medical Center, Omaha, NE 68198, United States.
Jacobs J Mol Transl Med. 2016 Feb;1(1). Epub 2015 Jun 9.
Self-assembly and aggregation of amyloid peptides, such as Aβ(1-40) and Aβ(1-42), lead to the development of Alzheimer disease and similar neurodegenerative disorders associated with protein aggregation. The structures of large aggregates, specifically fibrils, are well characterized. However, our understanding about the structure of oligomeric forms of amyloids is incomplete and needs to be expanded, particularly given the finding that oligomeric rather than fibrillar amyloid morphologies are neurotoxic. This lack of knowledge is primarily due to the existence of transient oligomeric forms that require the use of non-traditional approaches capable of probing transiently existing amyloid forms. We have recently developed the Single-Molecule Force Spectroscopy (SMFS) approach enabling us to probe dimeric forms of amyloids. These studies suggest that the assembly of amyloid proteins into dimers leads to extremely stabilized amyloids in non-native, misfolded states [1]. Herein, we applied the SMFS approach to probe amyloid trimers. We used the Aβ(14-23) segment of Aβ42 protein that is responsible for full-size protein aggregation. The dimerization of this peptide was recently characterized [2]. The dimeric form of Aβ (14-23) was assembled by the use of a tandem Aβ(14-23)-YNGK-Aβ(14-23), in which the YNGK motif between the two Aβ(14-23) monomers makes a β turn to form a hairpin loop with an antiparallel arrangement of Aβ(14-23) monomers[3]. The Aβ(14-23) monomer was tethered to the AFM tip, and trimers were formed by approaching the tip to the mica surface on which the Aβ(14-23)-YNGK-Aβ(14-23) dimer was immobilized via a polyethylene glycol tether. We identified trimers by rupture forces that were considerably larger than those for dimers. Models for the trimer assembly process are discussed.
淀粉样肽（如Aβ(1 - 40)和Aβ(1 - 42)）的自组装和聚集会导致阿尔茨海默病以及与蛋白质聚集相关的类似神经退行性疾病的发展。大型聚集体（特别是原纤维）的结构已得到充分表征。然而，我们对淀粉样蛋白寡聚体形式的结构了解并不完整，需要进一步拓展，尤其是考虑到寡聚而非原纤维状淀粉样形态具有神经毒性这一发现。这种知识的匮乏主要是由于存在瞬时寡聚体形式，这需要使用能够探测瞬时存在的淀粉样形式的非传统方法。我们最近开发了单分子力谱（SMFS）方法，使我们能够探测淀粉样蛋白的二聚体形式。这些研究表明，淀粉样蛋白组装成二聚体会导致处于非天然、错误折叠状态的极其稳定的淀粉样蛋白[1]。在此，我们应用SMFS方法探测淀粉样三聚体。我们使用了Aβ42蛋白中负责全长蛋白聚集的Aβ(14 - 23)片段。该肽的二聚化最近已得到表征[2]。Aβ(14 - 23)的二聚体形式通过使用串联的Aβ(14 - 23)-YNGK-Aβ(14 - 23)组装而成，其中两个Aβ(14 - 23)单体之间的YNGK基序形成一个β转角，与Aβ(14 - 23)单体的反平行排列形成一个发夹环[3]。将Aβ(14 - 23)单体连接到原子力显微镜（AFM）尖端，并通过将尖端靠近云母表面形成三聚体，在云母表面，Aβ(14 - 23)-YNGK-Aβ(14 - 23)二聚体通过聚乙二醇连接子固定。我们通过比二聚体大得多的断裂力识别出三聚体。文中讨论了三聚体组装过程的模型。
