Phu Mai-Jane, Hawbecker Sharon K, Narayanaswami Vasanthy
Lipid Biology in Health and Disease Research Group, Children's Hospital Oakland Research Institute, CA 94609, USA.
J Neurosci Res. 2005 Jun 15;80(6):877-86. doi: 10.1002/jnr.20503.
The potential neurotoxicity of soluble forms of amyloid beta peptide (Abeta) as a key factor in early pathogenesis of Alzheimer's disease is being recognized. In addition, there is growing evidence of the essential role of apolipoprotein E (apoE) in amyloid formation, although molecular details of apoE/Abeta interaction are poorly understood. We employed apoE C-terminal (CT) domain comprising residues 201-299 to identify binding location of Abeta(1-42) by fluorescence resonance energy transfer (FRET) and quenching analyses. Native tryptophan (Trp) residues in the apoE CT domain served as FRET donor, whereas N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine (AEDANS) covalently attached to a unique cysteine residue substituted at position 4 of Abeta(1-42) (AEDANS-F4C-Abeta(1-42)) served as FRET acceptor. Fluorescence analysis verified that the oligomerization behavior of AEDANS-F4C-Abeta(1-42) was not abrogated by covalent attachment of AEDANS and that apoE CT domain/AEDANS-F4C-Abeta(1-42) association results in formation of a soluble complex. A large decrease in Trp fluorescence emission was noted in mixtures containing apoE CT domain and AEDANS-F4C-Abeta(1-42), accompanied by appearance of sensitized fluorescence emission of AEDANS as a result of intermolecular FRET. An average distance of separation of 22.6 Angstroms between donors and acceptor was calculated. Fluorescence quenching by potassium iodide (KI) did not reveal significant differences in apoE CT domain Trp microenvironment in the absence or the presence of Abeta(1-42). A twofold increase in quenching constant was noted for KI quenching of AEDANS fluorescence emission in the presence of apoE CT domain, indicative of alterations in Abeta conformation upon interaction with apoE CT domain. We propose intermolecular FRET analysis as a discriminating approach to examine apoE/Abeta interaction, a potentially critical factor in early events involved in amyloid formation.
可溶性淀粉样β肽（Aβ）作为阿尔茨海默病早期发病机制中的关键因素，其潜在的神经毒性正在被人们所认识。此外，越来越多的证据表明载脂蛋白E（apoE）在淀粉样蛋白形成中起着重要作用，尽管apoE/Aβ相互作用的分子细节还知之甚少。我们采用包含201 - 299位残基的apoE C末端（CT）结构域，通过荧光共振能量转移（FRET）和猝灭分析来确定Aβ（1 - 42）的结合位置。apoE CT结构域中的天然色氨酸（Trp）残基作为FRET供体，而共价连接到Aβ（1 - 42）第4位取代的独特半胱氨酸残基上的N - （碘乙酰基） - N' - （5 - 磺基 - 1 - 萘基）乙二胺（AEDANS）（AEDANS - F4C - Aβ（1 - 42））作为FRET受体。荧光分析证实，AEDANS的共价连接并未消除AEDANS - F4C - Aβ（1 - 42）的寡聚化行为，并且apoE CT结构域/AEDANS - F4C - Aβ（1 - 42）的结合导致形成可溶性复合物。在含有apoE CT结构域和AEDANS - F4C - Aβ（1 - 42）的混合物中，观察到Trp荧光发射大幅下降，同时由于分子间FRET出现了AEDANS的敏化荧光发射。计算得出供体和受体之间的平均分离距离为22.6埃。碘化钾（KI）的荧光猝灭未显示在不存在或存在Aβ（1 - 42）的情况下apoE CT结构域Trp微环境有显著差异。在apoE CT结构域存在的情况下，KI对AEDANS荧光发射的猝灭常数增加了两倍，这表明Aβ与apoE CT结构域相互作用后构象发生了改变。我们提出分子间FRET分析作为一种鉴别方法来研究apoE/Aβ相互作用，这是淀粉样蛋白形成早期事件中一个潜在的关键因素。
