Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida-Shimoadachi-cho, Sakyo-ku, Kyoto 606-8501, Japan.
Biochemistry. 2012 Oct 16;51(41):8125-31. doi: 10.1021/bi300839u. Epub 2012 Oct 2.
The conversion of soluble, nontoxic amyloid β-proteins (Aβ) to aggregated, toxic forms rich in β-sheets is considered to be a key step in the development of Alzheimer's disease. Accumulating evidence suggests that lipid-protein interactions play a crucial role in the aggregation of amyloidogenic proteins like Aβ. Our group has previously reported that amyloid fibrils of Aβ formed on membranes containing clusters of GM1 ganglioside (M-fibrils) exhibit greater cytotoxicity than fibrils formed in aqueous solution (W-fibrils) [ Okada ( 2008 ) J. Mol. Biol. 382 , 1066 - 1074 ]. W-fibrils are considered to consist of in-register parallel β-sheets. However, the precise molecular structure of M-fibrils and force driving the formation of toxic fibrils remain unclear. In this study, we hypothesized that low-polarity environments provided by GM1 clusters drive the formation of toxic fibrils and compared the structure and cytotoxicity of W-fibrils, M-fibrils, and aggregates formed in a low-polarity solution mimicking membrane environments. First, we determined solvent conditions which mimic the polarity of raftlike membranes using Aβ-(1-40) labeled with the 7-diethylaminocoumarin-3-carbonyl dye. The polarity of a mixture of 80% 1,4-dioxane and 20% water (v/v) was found to be close to that of raftlike membranes. Aβ-(1-40) formed amyloid fibrils within several hours in 80% dioxane (D-fibrils) or in the presence of raftlike membranes, whereas a much longer incubation time was required for fibril formation in a conventional buffer. D-fibrils were morphologically similar to M-fibrils. Fourier-transform infrared spectroscopy suggested that M-fibrils and D-fibrils contained antiparallel β-sheets. These fibrils had greater surface hydrophobicity and exhibited significant toxicity against human neuroblastoma SH-SY5Y cells, whereas W-fibrils with less surface hydrophobicity were not cytotoxic. We concluded that ganglioside clusters mediate the formation of toxic amyloid fibrils of Aβ with an antiparallel β-sheet structure by providing less polar environments.
可溶性无毒淀粉样 β 蛋白（Aβ）转化为富含 β-折叠的聚集性毒性形式被认为是阿尔茨海默病发展的关键步骤。越来越多的证据表明，脂-蛋白相互作用在淀粉样蛋白样蛋白如 Aβ 的聚集中起着至关重要的作用。我们的研究小组之前曾报道过，在含有神经节苷脂 GM1 簇的膜上形成的 Aβ 淀粉样原纤维（M 原纤维）比在水溶液中形成的纤维（W 原纤维）表现出更高的细胞毒性[Okada（2008）J. Mol. Biol. 382, 1066-1074]。W 原纤维被认为由有序平行的 β-折叠组成。然而，M 原纤维的精确分子结构和形成毒性原纤维的驱动力仍不清楚。在这项研究中，我们假设 GM1 簇提供的低极性环境驱动毒性原纤维的形成，并比较了 W 原纤维、M 原纤维和在模拟膜环境的低极性溶液中形成的聚集体的结构和细胞毒性。首先，我们使用用 7-二乙氨基香豆素-3-羰基染料标记的 Aβ-（1-40）来确定模拟筏样膜极性的溶剂条件。发现 80%1,4-二氧杂环己烷和 20%水（v/v）混合物的极性接近于筏样膜的极性。Aβ-（1-40）在 80%二氧杂环己烷（D-原纤维）或存在筏样膜的情况下数小时内形成淀粉样原纤维，而在常规缓冲液中形成原纤维则需要更长的孵育时间。D-原纤维在形态上与 M 原纤维相似。傅里叶变换红外光谱表明，M 原纤维和 D-原纤维含有反平行 β-折叠。这些原纤维具有更大的表面疏水性，并对人神经母细胞瘤 SH-SY5Y 细胞表现出显著的毒性，而表面疏水性较小的 W 原纤维则没有细胞毒性。我们得出结论，神经节苷脂簇通过提供较少的极性环境来介导具有反平行 β-折叠结构的 Aβ毒性淀粉样原纤维的形成。
