Spontaneous aggregation and cytotoxicity of the β-amyloid Aβ1-40:: A kinetic model
Spontaneous aggregation and cytotoxicity of the β-amyloid Aβ1-40:: A kinetic model
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DOI：
10.1023/a:1023063626770
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发表时间：
2003-01-01
期刊：
影响因子：
--
通讯作者：
Epps, DE
中科院分区：
文献类型：
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作者：
Taylor, BM;Sarver, RW;Epps, DE
通过浊度、圆二色性、HPLC 和荧光偏振测量纤维化 β-淀粉样肽 Abeta(1-40) 自发聚集的时间依赖性。所有方法的结果都是可比的，并且与动力学模型最一致，其中肽首先缓慢形成活化的单体衍生物（AM），这是唯一能够通过四聚化启动线性聚集体形成的物种。针对残基 1-17 产生的抗 Abeta 抗体 6E10，浓度为 200-300 nM，显着延迟了 50 muM 淀粉样肽的聚集。针对残基17-24产生的抗Abeta抗体4G8在这方面活性低得多，而针对全长Abeta的C端残基39-43产生的抗体A162在这些浓度下完全没有活性。在聚集实验的同时，我们还测量了 Abeta(1-40) 诱导的对 SH-EP1 细胞和海马神经元的毒性的时间依赖性，通过 SYTOX Green 荧光、乳酸脱氢酶释放和半胱天冬酶激活进行评估。所有方法测得的细胞损伤程度均同时达到最大值，且该最大值与AM浓度一致。根据动力学方案，后者是唯一浓度达到最大值的瞬态肽种类。因此，Abeta(1-40) 的有毒物质很可能与负责原纤维形成成核的瞬时活化单体相同。这些结论应该为理解 Abeta(1-40) 的体外毒性和可能的体内毒性提供结构基础。
The time dependency of the spontaneous aggregation of the fibrillogenic beta-amyloid peptide, Abeta(1-40), was measured by turbidity, circular dichroism, HPLC, and fluorescence polarization. The results by all methods were comparable and they were most consistent with a kinetic model where the peptide first slowly forms an activated monomeric derivative ( AM), which is the only species able to initiate, by tetramerization, the formation of linear aggregates. The anti-Abeta antibody 6E10, raised against residues 1-17, at concentrations of 200-300 nM delayed significantly the aggregation of 50 muM amyloid peptide. The anti-Abeta antibody 4G8, raised against residues 17-24, was much less active in that respect, while the antibody A162, raised against the C-terminal residues 39-43 of the full-length Abeta was totally inactive at those concentrations. Concomitant with the aggregation experiments, we also measured the time dependency of the Abeta(1-40)-induced toxicity toward SH-EP1 cells and hippocampal neurons, evaluated by SYTOX Green fluorescence, lactate dehydrogenase release, and activation of caspases. The extent of cell damage measured by all methods reached a maximum at the same time and this maximum coincided with that of the concentration of AM. According to the kinetic scheme, the latter is the only transient peptide species whose concentration passes through a maximum. Thus, it appears that the toxic species of Abeta(1-40) is most likely the same transient activated monomer that is responsible for the nucleation of fibril formation. These conclusions should provide a structural basis for understanding the toxicity of Abeta(1-40) in vitro and possibly in vivo.