Department of Molecular Pharmacology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu-Chong-Zhi Road, Shanghai, 201203, China.
Anal Bioanal Chem. 2010 Mar;396(5):1745-54. doi: 10.1007/s00216-009-3420-6. Epub 2010 Feb 6.
Aβ(1-42) is the proteolytic cleavage product of cleavage of the amyloid precursor protein by β- and γ-secretases. The aggregation of Aβ(1-42) plays a causative role in the development of Alzheimer's disease. To lock Aβ(1-42) in a homogenous state, we embedded the Aβ(1-42) sequence in an unstructured region of Bcl-x(L). Both the N-terminus and the C-terminus of Aβ(1-42) were constrained in the disordered region, whereas the conjunction did not introduce any folding to Aβ(1-42) but maintained the sequence as a monomer in solution. With Bcl-x(L)-Aβ(42), we developed an enzyme-linked immunosorbent assay to compare the affinity of compounds for monomeric Aβ(1-42). Bcl-x(L)-Aβ(42) was coated on a microplate and this was followed by incubation with different concentrations of compounds. Compounds binding to Leu17-Val24 of Aβ(1-42) inhibited the interaction between Bcl-x(L)-Aβ(42) and antibody 4G8. The method can not only reproduce the activities of the reported Aβ(1-42) inhibitors such as dopamine, tannin, and morin but can also differentiate decoy compounds that do not bind to Aβ(1-42). Remarkably, using this method, we discovered a new inhibitor that binds to monomeric Aβ(1-42) and inhibits Aβ(1-42) fibril formation. As the structure of Bcl-x(L)-Aβ(42) monomer is stable in solution, the assay could be adapted for high-throughput screening with a series of antibodies that bind the different epitopes of Aβ(1-42). In addition, the monomeric form of the Aβ(1-42) sequence in Bcl-x(L)-Aβ(42) would also facilitate the identification of Aβ(1-42) binding partners by coimmunoprecipitation, cocrystallization, surface plasmon resonance technology, or the assay as described here.
β淀粉样蛋白（1-42）是淀粉样前体蛋白被β-和γ-分泌酶切割产生的蛋白水解产物。β淀粉样蛋白（1-42）的聚集在阿尔茨海默病的发展中起着致病作用。为了将β淀粉样蛋白（1-42）锁定在同一种状态，我们将β淀粉样蛋白（1-42）序列嵌入 Bcl-x(L)的无结构区域。β淀粉样蛋白（1-42）的 N 端和 C 端都被约束在无序区域，而连接并没有使β淀粉样蛋白（1-42）发生任何折叠，但保持了溶液中单体的序列。我们使用 Bcl-x(L)-Aβ(42) 开发了酶联免疫吸附测定法，以比较化合物对单体β淀粉样蛋白（1-42）的亲和力。Bcl-x(L)-Aβ(42) 被涂覆在微孔板上，然后与不同浓度的化合物孵育。与β淀粉样蛋白（1-42）的 Leu17-Val24 结合的化合物抑制了 Bcl-x(L)-Aβ(42)与抗体 4G8 之间的相互作用。该方法不仅可以重现报道的β淀粉样蛋白（1-42）抑制剂（如多巴胺、单宁和桑色素）的活性，还可以区分不与β淀粉样蛋白（1-42）结合的诱饵化合物。值得注意的是，使用这种方法，我们发现了一种新的抑制剂，它与单体β淀粉样蛋白（1-42）结合并抑制β淀粉样蛋白（1-42）纤维形成。由于 Bcl-x(L)-Aβ(42) 单体在溶液中的结构稳定，因此该测定法可以适应与结合β淀粉样蛋白（1-42）不同表位的一系列抗体进行高通量筛选。此外，Bcl-x(L)-Aβ(42) 中的β淀粉样蛋白（1-42）单体形式也将有助于通过免疫沉淀、共结晶、表面等离子体共振技术或此处所述的测定法鉴定β淀粉样蛋白（1-42）结合伴侣。
