BRD7核定位信号的结构分析和功能鉴定

Structural Analysis and Functional Identification of Nuclear Localization Signal Sequences in BRD7

目的:对BRD7的核定位信号进行预测、结构分析和功能鉴定,并考察其对BRD7亚细胞定位的影响。方法:通过生物信息学对BRD7的核定位信号进行预测和结构分析,然后利用绿色荧光蛋白(GFP)介导的直接荧光和间接免疫荧光定位方法分别对核定位信号的功能进行鉴定,并考察其对BRD7亚细胞定位的影响。结果:BRD7的65～96位氨基酸残基具有潜在核定位信号(NLS)的结构特征,该核定位信号包含3簇碱性氨基酸残基,可视为由2个紧密相邻、部分重叠的双向核靶序列NLS1和NLS2组成;并发现NLS及其构成上的NLS1和NLS2均具有介导异源蛋白GFP胞核定位的功能,从而证实BRD7的65～96位残基为BRD7功能性核定位信号所在区域,且单簇碱性氨基酸残基的缺失不足以破坏其核定位信号的功能;同时发现野生型BRD7呈胞核分布,而核定位信号缺失型BRD7主要呈胞浆分布。结论:BRD7的65～96位氨基酸残基为BRD7功能性核定位信号所在区域,在BRD7胞核分布模式中发挥了十分重要的作用。

Objective： To structurally analyse and functionally identify the nuclear localization signal（NLS） in BBD7 and then study its effect on the subcellular localization of BRD7. Methods： Bioinformatics was performed to predict and anslyse the nuclear localization signal sequenees（NLSs） in BBD7, then green fluorescent protein（GFP） direct fluorescence and indirect immunofluoreseenee assays were used to identify the function of the NLSs and the effect on the subcellular localization of BBDT. Results： The region from amino acid 65 to 96 in BBD7 was characteristic of putative nuclear localization signal sequence and contained three clusters of base amino acid residues. It was viewed to consist of two bipartite nuclear targeting sequences, NLS1 and NLS2, which were tightly linked and extremely overlapped. It was also shown that both the entire NLS and the two bipartite nudear targeting sequences, NLS1 and NLS2, could respectively determine the nuclear import of GFP, which supported that the region from aa 65 to 96 in BRD7 was a functional nudear localization signal and the deletion of a duster of base residues was insufficient to demolish the function of the NLS. The most important was that wild BRD7 localized in nudeus, whereas NLS-deleted BRD7 shifted the nuclear localization to be mostly in cytoplasm. Conclusion： The amino acid region from 65 to 96 is a functional NLS in BRD7 and it is an essential motif affecting BRD7 nuclear distribution.