β淀粉样蛋白突变体的寡聚及插膜能力的鉴定

Characterization of the Behavior of β-Amyloid Peptide Mutants in the Processes of Oligomerization and Membrane Insertion

β-淀粉样蛋白（β-amyloid peptide,Aβ）的插膜与寡聚是导致阿尔茨海默症（Alzheimer disease,AD）发病的重要事件。已有研究证明,Aβ氨基酸序列29~36与1~28依靠＂核心疏水簇＂（central hydrophobic cluster,CHC）的作用形成一个稳定的β-发夹结构,Aβ1-40/Aβ1-42的插膜与寡聚需要作用于序列37~40/37~42从而解除序列29~36与N-端之间的结合,但各基元序列如何互作从而贡献出Aβ的寡聚及插膜行为仍不清楚。本文主要研究Aβ1-28、Aβ1-36、Aβ1-40、Aβ1-42、Aβ11-42、Aβ17-42等突变体的寡聚和插膜能力,并探讨各基元序列（motif）在突变体插膜与寡聚过程中的相互作用。Western印迹、硫黄素T荧光分析、透射电镜等实验检测寡聚能力,模型膜实验比较插膜能力。结果显示：与Aβ1-28及Aβ1-36相比,Aβ1-42、Aβ11-42及Aβ17-42均具有较强的寡聚及插膜能力,说明C-端序列37~42在Aβ寡聚及插膜过程中具有重要的起始作用;Aβ1-42及Aβ1-40可以形成原纤维及纤维,但Aβ11-42、Aβ17-42却不能,这显示序列1~17可以稳定纤维结构。Aβ1-28及Aβ1-36插膜及寡聚能力弱,暗示这两个突变体可能形成了不容易插膜且不容易寡聚的自身稳定结构。上述结果证明,Aβ蛋白C-端是诱导插膜与寡聚的主因,N-端可以稳定长纤维,但对插膜和寡聚的影响并不大,中间肽段很可能形成一个自身稳定的区域,这在一定程度上解释Aβ基元序列的相互作用,但具体氨基酸互作分子机制及抑制方法还需进一步研究。

The oligomerization and membrane insertion of beta-amyloid peptide（Aβ） is an important event in Alzheimer disease pathogenesis.It has been proved that amino acid 29 ~ 36 and 1 ~ 28 depending on the role of the ＂core hydrophobic cluster＂form a stable beta hairpin structure,a common initiating event appears to be shared by the soluble oligomerization and membrane insertion,i.e.overcoming the interaction between 29 ~ 36 and 1 ~ 28 through the action of 37-40/42 albeit via different mechanisms.However,how the motifs within Aβ sequence contribute to oligomerization and membrane insertion is still not clear.In order to reveal a complex interplay among the short motifs within Aβ,this paper mainly discussed the oligomerization and membrane insertion ability of Aβ1-28,Aβ1-36,Aβ1-40,Aβ1-42,Aβ11-42 and Aβ17-42.Th T fluorescence detection,Western blotting,transmission electron microscopy experiments were carried out to detect the six Aβ truncation variants＇ oligomerization ability.And the model membrane experiments were carried out to detect their membrane insertion ability.The experiments showed that compared to two C-terminal truncated peptides i.e.Aβ1-28 and Aβ1-36,Aβ11-42,Aβ17-42 and Aβ1-42 all had a stronger ability to insert membrane and form oligomers.This indicated that amino acids 37 ~ 42 played an important role in the process of Aβ oligomerization and membrane insertion;Aβ1-42 and Aβ1-40 can form protofibril and fiber,but Aβ11-42 and Aβ17-42 can not,which reflected that amino acid 1 ~ 17 was essential for Aβ fibrillation.Aβ1-28 and Aβ1-36 were weak in oligomerization and membrane insertion,implying they form a relatively stable structure.The above results showed Aβ C-terminal initiating membrane insertion and oligomerization.The C-terminal has the function of stabilizing long fibers.Intermediate peptides are likely to form a stable region themselves.To a certain extent,this work explains the interplay of motifs within Aβ in the membrane insertion and oligomerization.But the molecular mechanism of the interaction between the specific amino acids and the inhibition method need to be further studied.