Leibniz-Institut für Molekulare Pharmakologie, Robert-Rössle Strasse 10, 13125 BerlinBuch, Germany.
Chembiochem. 2011 Feb 11;12(3):407-23. doi: 10.1002/cbic.201000602. Epub 2011 Jan 10.
The structures of oligomeric intermediate states in the aggregation process of Alzheimer's disease β-amyloid peptides have been the subject of debate for many years. Bacterial inclusion bodies contain large amounts of small heat shock proteins (sHSPs), which are highly homologous to those found in the plaques of the brains of Alzheimer's disease patients. sHSPs break down amyloid fibril structure in vitro and induce oligomeric assemblies. Prokaryotic protein overexpression thus mimics the conditions encountered in the cell under stress and allows the structures of Aβ aggregation intermediate states to be investigated under native-like conditions, which is not otherwise technically possible. We show that IB40/IB42 fulfil all the requirements to be classified as amyloids: they seed fibril growth, are Congo red positive and show characteristic β-sheet-rich CD spectra. However, IB40 and IB42 are much less stable than fibrils formed in vitro and contain significant amounts of non-β-sheet regions, as seen from FTIR studies. Quantitative analyses of solution-state NMR H/D exchange rates show that the hydrophobic cores involving residues V18-F19-F20 adopt β-sheet conformations, whereas the C termini adopt α-helical coiled-coil structures. In the past, an α-helical intermediate-state structure has been postulated, but could not be verified experimentally. In agreement with the current literature, in which Aβ oligomers are described as the most toxic state of the peptides, we find that IB42 contains SDS-resistant oligomers that are more neurotoxic than Aβ42 fibrils. E. coli inclusion bodies formed by the Alzheimer's disease β-amyloid peptides Aβ40 and Aβ42 thus behave structurally like amyloid aggregation intermediate states and open the possibility of studying amyloids in a native-like, cellular environment.
阿尔茨海默病β-淀粉样肽聚集过程中寡聚中间体状态的结构多年来一直存在争议。细菌包涵体含有大量的小分子热休克蛋白（sHSPs），与阿尔茨海默病患者大脑斑块中发现的高度同源。sHSPs 在体外破坏淀粉样纤维结构并诱导寡聚组装。因此，原核蛋白的过表达模拟了应激下细胞中遇到的条件，并允许在类似天然的条件下研究 Aβ 聚集中间体状态的结构，否则在技术上是不可能的。我们表明，IB40/IB42 满足被分类为淀粉样蛋白的所有要求：它们引发纤维生长，刚果红阳性，并显示出特征性富含β-折叠的 CD 光谱。然而，与体外形成的纤维相比，IB40 和 IB42 稳定性要差得多，FTIR 研究表明它们含有大量非β-折叠区域。溶液状态 NMR H/D 交换率的定量分析表明，涉及残基 V18-F19-F20 的疏水区采用β-折叠构象，而 C 末端采用α-螺旋卷曲螺旋结构。过去，已经提出了一种α-螺旋中间态结构，但无法通过实验验证。与当前文献一致，其中将 Aβ 低聚物描述为肽的最毒状态，我们发现 IB42 含有 SDS 抗性低聚物，比 Aβ42 纤维更具神经毒性。因此，由阿尔茨海默病β-淀粉样肽 Aβ40 和 Aβ42 形成的大肠杆菌包涵体在结构上表现得像淀粉样聚集中间态，并为在类似天然的、细胞环境中研究淀粉样蛋白提供了可能性。
