Department of Chemistry, Binghamton University, the State University of New York, Vestal, New York 13850, United States.
Biochemistry. 2024 Oct 15;63(20):2557-2564. doi: 10.1021/acs.biochem.4c00395. Epub 2024 Sep 30.
Molecular-level structural polymorphisms of β-amyloid (Aβ) fibrils have recently been recognized as pathologically significant. High-resolution solid-state nuclear magnetic resonance (ssNMR) spectroscopy has been utilized to study these structural polymorphisms, particularly in ex-vivo fibrils seeded from amyloid extracts of post-mortem brain tissues of Alzheimer's disease (AD) patients. One unaddressed question in current ex-vivo seeding protocol is whether fibrillation from exogenous monomeric Aβ peptides, added to the extracted seeds, can be quantitatively suppressed. Addressing this issue is critical because uncontrolled fibrillation could introduce biased molecular structural polymorphisms in the resulting fibrils. Here, we present a workflow to optimize the key parameters of ex-vivo seeding protocols, focusing on the quantification of amyloid extraction and the selection of exogenous monomeric Aβ concentrations to minimize nonseeded fibrillation. We validate this workflow using three structurally different 40-residue Aβ (Aβ) fibrillar seeds, demonstrating their ability to propagate their structural features to exogenous wild-type Aβ.
β-淀粉样蛋白（Aβ）纤维的分子水平结构多态性最近被认为具有病理学意义。高分辨率固态核磁共振（ssNMR）光谱已被用于研究这些结构多态性，特别是在从阿尔茨海默病（AD）患者死后脑组织的淀粉样提取物中接种的体外纤维中。目前外源性接种方案中一个未解决的问题是，添加到提取种子中的外源性单体 Aβ 肽的纤维形成是否可以被定量抑制。解决这个问题至关重要，因为不受控制的纤维形成可能会导致产生的纤维中存在有偏差的分子结构多态性。在这里，我们提出了一个优化外源性接种方案关键参数的工作流程，重点是定量测定淀粉样蛋白的提取和选择外源性单体 Aβ 浓度，以最小化未接种的纤维形成。我们使用三种结构不同的 40 个残基 Aβ（Aβ）纤维状种子验证了该工作流程，证明了它们能够将其结构特征传播到外源性野生型 Aβ。
