小分子伴侣对淀粉样蛋白β聚集抑制作用研究

Suppression of amyloidβaggregation with minichaperone

淀粉样蛋白β(Aβ)的积累和聚集在阿尔茨海默症的致病机理研究中是非常关键的,因而开发能有效抑制Aβ聚集的新型抑制剂具有重要的意义。来源于大肠杆菌分子伴侣GroEL/GroES系统的小分子伴侣(GroEL顶端结构域191-345位残基)可以通过疏水相互作用与目标蛋白结合,具有生物相容性好等特点,是一种潜在的Aβ聚集抑制剂。通过硫代硫磺素T荧光光谱、透射电镜、凝胶过滤色谱分析和细胞毒性实验详细研究了小分子伴侣与Aβ42的相互作用,并与姜黄素、表没食子儿茶素没食子酸酯和槲皮素等多酚类抑制剂的作用进行比较。结果表明小分子伴侣不仅降低了Aβ42的聚集程度,同时也改变了Aβ42的聚集路径,使得其聚集体分子量和纤维形貌发生了显著变化。相较于对照组,添加小分子伴侣后使得Aβ42溶液原位ThT荧光响应值下降了72%,形成的纤维长度从数百纳米下降到小于50 nm,SH-SY5Y的细胞存活率提升了20%。因此,小分子伴侣能有效阻止Aβ纤维聚集体形成,降低其细胞毒性,具有良好的应用前景。

Alzheimer’s disease(AD)is one of the most common neurodegenerative diseases that the death of neuron cells causes severe memory loss and cognitive decline.The accumulation and aggregation of amyloid beta protein(Aβ)is closely related to AD pathogenesis.Therefore,it is meaningful to develop novel suppressors that can effectively suppress Aβaggregation.Minichaperone derived from apical domain of E.coli chaperone GroEL(residues 191-345)is a potential suppressor for Aβaggregation,which can bind target proteins through hydrophobic interactions with good biocompatibility.A series of biochemical and biophysical experiments(thioflavin T(ThT)fluorescence spectra,transmission electron microscopy(TEM),gel filtration chromatography analysis(ana-SEC),and cytotoxicity assay)were performed to investigate the interaction between minichaperone and Aβ42.Meanwhile,the effects of polyphenol suppressors such as curcumin,epigallocatechin gallate(EGCG)and quercetin were also studied.Minichaperone can effectively suppress the formation of Aβ42 fibrils.The aggregation suppression is achieved by reducing the aggregation level of Aβ42 and shifting the on-pathway aggregates to off-pathway aggregates.The molecular weights of aggregates and fibrils morphology change significantly.Compared to the control group,addition of the minichaperone decreases in situ ThT fluorescence of Aβ42 solution up to 72%after 16 h of aging,shortens the length of Aβ42 fibril from>200 nm to<50 nm after 24 h of aging,and increases SH-SY5 Y cell viability by more than 20%.Therefore,minichaperone presents a promising therapeutic application as a suppressor of Aβaggregation by prevention of amyloid fibril formation and reduction of Aβcytotoxicity.