Silvers Robert, Colvin Michael T, Frederick Kendra K, Jacavone Angela C, Lindquist Susan, Linse Sara, Griffin Robert G
Department of Chemistry and Francis Bitter Magnet Laboratory, Massachusetts Institute of Technology , Cambridge, Massachusetts 02139, United States.
Whitehead Institute for Biomedical Research , Nine Cambridge Center, Cambridge, Massachusetts 02142, United States.
Biochemistry. 2017 Sep 12;56(36):4850-4859. doi: 10.1021/acs.biochem.7b00729. Epub 2017 Aug 30.
A mechanistic understanding of Aβ aggregation and high-resolution structures of Aβ fibrils and oligomers are vital to elucidating relevant details of neurodegeneration in Alzheimer's disease, which will facilitate the rational design of diagnostic and therapeutic protocols. The most detailed and reproducible insights into structure and kinetics have been achieved using Aβ peptides produced by recombinant expression, which results in an additional methionine at the N-terminus. While the length of the C-terminus is well established to have a profound impact on the peptide's aggregation propensity, structure, and neurotoxicity, the impact of the N-terminal methionine on the aggregation pathways and structure is unclear. For this reason, we have developed a protocol to produce recombinant Aβ, sans the N-terminal methionine, using an N-terminal small ubiquitin-like modifier-Aβ fusion protein in reasonable yield, with which we compared aggregation kinetics with Aβ containing the additional methionine residue. The data revealed that Aβ and Aβ aggregate with similar rates and by the same mechanism, in which the generation of new aggregates is dominated by secondary nucleation of monomers on the surface of fibrils. We also recorded magic angle spinning nuclear magnetic resonance spectra that demonstrated that excellent spectral resolution is maintained with both Aβ and Aβ and that the chemical shifts are virtually identical in dipolar recoupling experiments that provide information about rigid residues. Collectively, these results indicate that the structure of the fibril core is unaffected by N-terminal methionine. This is consistent with the recent structures of Aβ in which M0 is located at the terminus of a disordered 14-amino acid N-terminal tail.
对β-淀粉样蛋白（Aβ）聚集的机制理解以及Aβ原纤维和寡聚体的高分辨率结构对于阐明阿尔茨海默病神经退行性变的相关细节至关重要，这将有助于合理设计诊断和治疗方案。使用重组表达产生的Aβ肽获得了关于结构和动力学最详细且可重复的见解，这导致在N端额外出现一个甲硫氨酸。虽然C端长度对肽的聚集倾向、结构和神经毒性有深远影响已得到充分证实，但N端甲硫氨酸对聚集途径和结构的影响尚不清楚。因此，我们开发了一种方案，使用N端小泛素样修饰物-Aβ融合蛋白以合理产率生产不含N端甲硫氨酸的重组Aβ，并将其聚集动力学与含有额外甲硫氨酸残基的Aβ进行比较。数据显示，Aβ和Aβ以相似的速率并通过相同的机制聚集，其中新聚集体的产生主要由原纤维表面单体的二次成核主导。我们还记录了魔角旋转核磁共振谱，结果表明Aβ和Aβ都保持了出色的谱分辨率，并且在提供有关刚性残基信息的偶极重耦合实验中化学位移几乎相同。总体而言，这些结果表明原纤维核心结构不受N端甲硫氨酸的影响。这与最近的Aβ结构一致，其中M0位于无序的14个氨基酸N端尾巴的末端。
