物理化学学报

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Aβ40和hIAPP在溶液和表面的成核与交叉成核聚集行为

刘苗苗, 王文娟, 郝秀萍, 董晓燕   

  1. 天津大学化工学院生物工程系, 系统生物工程教育部重点实验室, 天津 300072
  • 收稿日期:2020-02-20 修回日期:2020-04-06 录用日期:2020-04-09 发布日期:2020-04-20
  • 通讯作者: 董晓燕 E-mail:d_xy@tju.edu.cn
  • 基金资助:
    国家自然科学基金(21978207)资助项目

Seeding and Cross-Seeding Aggregations of Aβ40 and hIAPP in Solution and on Surface

Miaomiao Liu, Wenjuan Wang, Xiuping Hao, Xiaoyan Dong   

  1. Key Laboratory of Systems Bioengineering of the Ministry of Education, Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300354, P. R. China
  • Received:2020-02-20 Revised:2020-04-06 Accepted:2020-04-09 Published:2020-04-20
  • Supported by:
    The project was supported by the National Natural Science Foundation of China (21978207).

摘要: 阿尔茨海默氏病(AD)和2型糖尿病(T2DM)是常见的由蛋白质错误折叠引起的疾病,作为与此二者相关的致病蛋白,淀粉样β蛋白(Aβ)和人胰岛淀粉样多肽(hIAPP)的交叉聚集行为暗示了AD和T2DM的相关性。然而,Aβ和hIAPP在体内的交叉聚集过程尚不明确。为了更好地模拟体内环境特征,即同时存在不同形式的淀粉样蛋白聚集体,且少量的聚集体附着在血管壁上会成为聚集过程的种子,本文以硫代黄素T荧光测定,原子力显微镜,圆二色光谱,石英晶体微天平以及MTT法作为研究手段,探究了Aβ和hIAPP在溶液和固体表面的成核与交叉成核聚集行为。结果表明,少量的Aβ40和hIAPP种子(单体浓度的1/50)即可显著改变异源聚集的聚集路径,形成具有不同形态且含有更多β-折叠结构的异源聚集体,导致更高的细胞毒性。溶液和固体表面上的结果均证明异源成核聚集效率低于同源聚集,且异源聚集的特征很大程度上取决于种子类型。此外,不同于溶液中所得结果,hIAPP种子在固体表面的交叉成核聚集效率显著高于Aβ40种子,证明了界面性质对交叉聚集过程的影响。这些结论对于理解淀粉样蛋白交叉聚集过程具有重要意义。

关键词: 淀粉样β蛋白, 人胰岛淀粉样多肽, 淀粉样蛋白聚集, 交叉成核, 界面

Abstract: Alzheimer’s disease (AD) and type 2 diabetes mellitus (T2DM), common incurable diseases caused by protein misfolding, have shown extensive correlation with each other via cross-aggregation between their related pathogenic peptide, amyloid β protein (Aβ) and human islet amyloid polypeptide (hIAPP), respectively. However, little is known about how these two peptides affect the cross-amyloid aggregation process in vivo. To better simulate the intracorporal environment, where different forms of amyloid aggregates co-exist and very few aggregates probably attach to the vessel wall as seeds, herein, we study the seeded-aggregation of Aβ and hIAPP in the presence of homogeneous or heterogeneous seeds, both in solution and on the solid surface, with different monomer and seed concentrations. In this study, Thioflavin T (ThT) fluorescence assay, atomic force microscopy (AFM), and far-UV circular dichroism (CD) were performed to investigate the aggregation process in solution. Moreover, the binding of monomers with seeds on solid surface was detected by quartz crystal microbalance with dissipation (QCM-D). The 3-(4,5-dime-thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays with human neuroblastoma cells (SH-SY5Y) were finally used to test the cytotoxicity caused by the aggregates. Series of analyses con?rmed that a small amount of Aβ40 or hIAPP seeds (1/50 of the monomers in solution) significantly changed the aggregation pathway, forming heterogeneous aggregates with different morphologies and increased β-sheet structures. MTT result showed that the heterogeneous aggregates obtained with Aβ40 and hIAPP seeding reduced the cell viability to 70.5% and 74.4%, respectively, both causing higher cytotoxicity than homogeneous aggregates (82.9% and 76.5%, respectively). The results in solution and on the solid surface both prove that Aβ40 and hIAPP seeds can not only induce rapid aggregation of their homogeneous monomers but also promote the heterogeneous monomers to aggregate, but monomer–heterogeneous seed binding efficiency is lower than that between homogeneous species. The differences in seeding and cross-seeding ability of Aβ40 and hIAPP indicate the barriers depended on the sequence similarity and structural compatibility between different amyloid aggregates. In the case of heterogeneous aggregation, aggregation features largely depend on the seeds. Furthermore, hIAPP seeds exhibited higher cross-seeding efficiency than Aβ40 seeds on the solid surface, which is different from the result in solution where Aβ40 seeds indicating the influence of interfacial properties on aggregation process. This finding would give a deep understanding of the cross-seeding aggregation process and we hope that this work will stimulate more research to explore all possible fundamental and practical aspects of amyloid cross-seeding.

Key words: Amyloid β protein, Human islet amyloid polypeptide, Amyloid aggregation, Cross-seeding, Interface

MSC2000: 

  • O641