论文部分内容阅读
目的:探讨驱动蛋白KIF4A调节肺耐药相关蛋白LRP在细胞内分布的作用机制,及其在肿瘤耐药过程中的作用。创新点:首次发现驱动蛋白KIF4A的C端区与肺耐药相关蛋白LRP的N端区结合,且KIF4A调节LRP在胞内分布依赖KIF4A的N端马达结构域。方法:应用免疫共沉淀和免疫荧光技术检测KIF4A与LRP的结合。根据KIF4A及LRP蛋白结构,构建绿色荧光蛋白(GFP)融合KIF4A截短突变质粒及Flag融合LRP截短突变质粒,免疫沉淀分析KIF4A与LRP相互作用区域。通过RNA干涉(RNAi)内源KIF4A,外源转入KIF4A截短突变体质粒,检测LRP在胞内分布。结论:本实验中免疫沉淀及免疫荧光结果显示,KIF4A与LRP结合,且在微管上有共定位(图1)。截短突变体免疫沉淀实验结果表明,KIF4A的C端尾部结构域与LRP的N端区结合(图2),RNAi敲降内源KIF4A表达导致LRP聚集在细胞核周围(图3),外源表达全长KIF4A可恢复LRP在胞内弥散状定位,但外源表达KIF4A的C端或N端截短突变无法恢复LRP的胞内定位,仍聚集在核周区域(图4)。综上所述,驱动蛋白KIF4A可与LRP结合,并调节LRP在胞内定位,KIF4A的C端尾部结构域与LRP结合,N端马达结构域促进LRP在胞内运输,二者缺一不可。
AIM: To investigate the mechanism of KIF4A regulating the intracellular distribution of lung-resistance-associated protein LRP and its role in the drug resistance of tumor. Innovative point: It was first discovered that the C-terminal region of kinesin KIF4A binds to the N-terminal region of lung resistance-related protein LRP and that KIF4A regulates the intracellular distribution of LRP dependent on the N-terminal motor domain of KIF4A. Methods: Co-immunoprecipitation and immunofluorescence were used to detect the binding of KIF4A to LRP. According to the structure of KIF4A and LRP proteins, a green fluorescent protein (GFP) fusion KIF4A truncated mutant plasmid and a Flag fusion LRP truncated mutant plasmid were constructed, and the interaction between KIF4A and LRP was analyzed by immunoprecipitation. Endogenous KIF4A was transfected into KIF4A mutant by RNA interference (RNAi), and LRP was detected intracellularly. CONCLUSIONS: Immunoprecipitation and immunofluorescence results in this experiment show that KIF4A binds to LRP and co-locates on microtubules (Figure 1). Immunoprecipitation experiments with truncated mutants showed that the C-terminal tail domain of KIF4A binds to the N-terminal region of LRP (Figure 2), and that RNAi knockdown endogenous KIF4A expression causes LRP to accumulate around the nucleus (Figure 3), exogenous Full-length KIF4A restored intracellular localization of LRP, but C-terminal or N-terminal truncation of exogenously expressed KIF4A failed to restore intracellular localization of LRP and remained clustered in the perinuclear region (Figure 4). In summary, kinesin KIF4A binds to LRP and regulates the localization of LRP in the cell. The C-terminal tail domain of KIF4A binds to LRP, and the N-terminal motor domain promotes intracellular transport of LRP, both of which are indispensable.