Alvarez A, Opazo C, Alarcón R, Garrido J, Inestrosa N C
Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, Chile.
J Mol Biol. 1997 Sep 26;272(3):348-61. doi: 10.1006/jmbi.1997.1245.
Acetylcholinesterase (AChE), an enzyme involved in the hydrolysis of the neurotransmitter acetylcholine, consistently colocalizes with the amyloid deposits characteristic of Alzheimer's disease and may contribute to the generation of amyloid proteins and/or physically affect fibril assembly. In order to identify the structural domains of the amyloid-beta-peptide (Abeta) involved in the aggregation induced by AChE, we have studied the effect of this cholinergic enzyme on Abeta peptide fragments of different sizes. AChE enhanced the aggregation of the Abeta(12-28) and Abeta(25-35) peptides but not of the Abeta(1-16) fragment. The inductive effect of AChE on the aggregation of Abeta(12-28) was abolished by the presence of either Abeta(1-16) or Abeta(9-21). The effect of the enzyme was also analysed using two different mutant fragments, possessing a low and the other a high capacity for fibrillogenesis. The fragments used were Abeta(12-28)Val18-->Ala and Abeta(12-28)Glu22-->Gln, respectively. AChE was able to promote the aggregation of these fragments in a very specific way and both mutant peptides were able to form amyloid fibrils, as revealed by negative staining under the electron microscope. Binding assays indicated that AChE was bound to Abeta(12-28), as well as to the Abeta(1-16) peptide. AChE was seen to form strong complexes with the Abeta(12-28) fibrils as such complexes stained positively for both thioflavine-T and AChE activity, were resistant to high ionic strength treatment, and were partially sensitive to detergents, suggesting that hydrophobic interactions may play a role in the stabilization of the AChE-Abeta complex. Our results suggest that such amyloid-AChE complexes are formed when AChE interacts with the growing amyloid fibrils and accelerates the assembly of Abeta peptides. This is consistent with the fact that AChE is known to be present within Abeta deposits including the pre-amyloid diffuse and mature senile plaques found in Alzheimer's brain.
乙酰胆碱酯酶（AChE）是一种参与神经递质乙酰胆碱水解的酶，它始终与阿尔茨海默病特有的淀粉样沉积物共定位，可能有助于淀粉样蛋白的产生和/或对纤维组装产生物理影响。为了确定淀粉样β肽（Aβ）中参与由AChE诱导的聚集的结构域，我们研究了这种胆碱能酶对不同大小的Aβ肽片段的影响。AChE增强了Aβ（12 - 28）和Aβ（25 - 35）肽的聚集，但对Aβ（1 - 16）片段没有影响。Aβ（1 - 16）或Aβ（9 - 21）的存在消除了AChE对Aβ（12 - 28）聚集的诱导作用。还使用了两种不同的突变片段分析了该酶的作用，其中一个具有低原纤维形成能力，另一个具有高原纤维形成能力。所使用的片段分别是Aβ（12 - 28）Val18→Ala和Aβ（12 - 28）Glu22→Gln。AChE能够以非常特定的方式促进这些片段的聚集，并且如电子显微镜下的负染色所示，两种突变肽都能够形成淀粉样纤维。结合试验表明，AChE与Aβ（12 - 28）以及Aβ（1 - 16）肽结合。可以看到AChE与Aβ（12 - 28）纤维形成强复合物，因为这种复合物对硫黄素 - T和AChE活性均呈阳性染色，对高离子强度处理具有抗性，并且对去污剂部分敏感，这表明疏水相互作用可能在AChE - Aβ复合物的稳定中起作用。我们的结果表明，当AChE与正在生长的淀粉样纤维相互作用并加速Aβ肽的组装时，会形成这种淀粉样 - AChE复合物。这与已知AChE存在于Aβ沉积物中这一事实一致，这些沉积物包括在阿尔茨海默病大脑中发现的淀粉样前体弥漫性和成熟老年斑。
