A plant cell model of polyglutamine aggregation: Identification and characterisation of macromolecular and small-molecule anti-protein aggregation activity in vivo.
A plant cell model of polyglutamine aggregation: Identification and characterisation of macromolecular and small-molecule anti-protein aggregation activity in vivo.
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DOI：
10.1016/j.jbiotec.2015.05.003
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发表时间：
2015-08
影响因子：
4.1
通讯作者：
Guobao Liu;Yueming Hu;A. Tunnacliffe;Yizhi Zheng
中科院分区：
文献类型：
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作者：
Guobao Liu;Yueming Hu;A. Tunnacliffe;Yizhi Zheng
体外研究表明，植物和无脊椎动物的LEA蛋白在水分胁迫条件下保护和稳定其他蛋白质，这表明在胁迫耐受中发挥了作用。然而，关于LEA蛋白在整个植物或植物细胞中的功能，特别是关于它们的抗聚集活性的信息很少。为了解决这个问题，我们在烟草BY-2悬浮细胞中表达了一种基于导致亨廷顿病(HD)的容易聚集的蛋白质。在HD中，Huntingtin(Htt)蛋白中的多聚谷氨酰胺(PolyQ)的异常长片段导致Htt片段在细胞内聚集。我们构建了稳定转化的表达增强型绿色荧光蛋白(EGFP)-HttQ23或EGFP-HttQ52融合蛋白(编码23或52个谷氨酰胺残基，分别属于正常和疾病状态)的BY-2细胞系，并以EGFP为对照。EGFP-HttQ52蛋白聚集在转化烟草细胞的细胞质中，表现出缓慢的生长动力学；相反，EGFP-HttQ23和EGFP没有形成聚集体，表达这些结构的细胞正常生长。为了测试LEA蛋白对植物细胞蛋白质聚集的影响，我们构建了同时表达EGFP-HttQ52和LEA蛋白(PM1、PM18、ZLDE-2或AavLEA1)或SHSP(PM31)的细胞系。其中，AavLEA1和PM31减少了细胞内EGFP-HttQ52的聚集，减轻了相关的生长抑制，而PM18和ZLDE-2部分恢复了细胞的生长速度。用多酚表没食子儿茶素没食子酸酯(EGCG)处理表达EGFP-HttQ52的BY-2细胞，也减少了EGFP-HttQ52的聚集，提高了细胞生长速度。因此，EGFP-HttQ52细胞系具有表征植物细胞中蛋白质聚集的大分子和小分子抑制物的潜力。
In vitro studies have shown that LEA proteins from plants and invertebrates protect and stabilise other proteins under conditions of water stress, suggesting a role in stress tolerance. However, there is little information on LEA protein function in whole plants or plant cells, particularly with respect to their anti-aggregation activity. To address this, we expressed in tobacco BY-2 suspension cells an aggregation-prone protein based on that responsible for Huntington's disease (HD). In HD, abnormally long stretches of polyglutamine (polyQ) in huntingtin (Htt) protein cause aggregation of Htt fragments within cells. We constructed stably transformed BY-2 cell lines expressing enhanced green fluorescent protein (EGFP)-HttQ23 or EGFP-HttQ52 fusion proteins (encoding 23 or 52 glutamine residues, pertaining to the normal and disease states, respectively), as well as an EGFP control. EGFP-HttQ52 protein aggregated in the cytoplasm of transformed tobacco cells, which showed slow growth kinetics; in contrast, EGFP-HttQ23 or EGFP did not form aggregates and cells expressing these constructs grew normally. To test the effect of LEA proteins on protein aggregation in plant cells, we constructed cell lines expressing both EGFP-HttQ52 and LEA proteins (PM1, PM18, ZLDE-2 or AavLEA1) or a sHSP (PM31). Of these, AavLEA1 and PM31 reduced intracellular EGFP-HttQ52 aggregation and alleviated the associated growth inhibition, while PM18 and ZLDE-2 partially restored growth rates. Treatment of EGFP-HttQ52-expressing BY2 cells with the polyphenol epigallocatechin-3-gallate (EGCG) also reduced EGFP-HttQ52 aggregation and improved cell growth rate. The EGFP-HttQ52 cell line therefore has potential for characterising both macromolecular and small molecule inhibitors of protein aggregation in plant cells.