核受体辅活化蛋白7与自噬体蛋白LC3的相互作用

Nuclear receptor co-activator 7 interacts with the autophagosomal protein LC3

目的:探索核受体辅活化蛋白7(nuclear receptor co-activator 7,NCOA7)与微管相关蛋白轻链3(microtubule-associated protein light chain 3,LC3)的相互作用,并确定其结合位点。方法:在胃癌AGS细胞中行质谱分析,在HEK293细胞中行GST-LC3沉淀实验,分别检测两种细胞中LC3结合蛋白;采用分子克隆法构建NCOA7不同截短体和点突变体;免疫印迹法检测NCOA7的截短体ΔN462,ΔN650,ΔN650-I749/750A质粒蛋白的表达水平;GST-LC3与NCOA7突变体的沉淀实验鉴定NCOA7与LC3的结合位点。结果:质谱分析和GST-LC3沉淀实验结果表明,NCOA7是LC3结合蛋白。免疫印迹结果显示,NCOA7不同截短体和点突变体质粒蛋白均有表达。GST-LC3与NCOA7突变体的沉淀结果显示,NCOA7 N端WEDL基序和C端WEII基序是LC3结合位点。WEII基序与LC3结合受到NCOA7的651~720位氨基酸结构抑制,是一个可调节的LC3结合位点。结论:NCOA7是LC3结合蛋白,其N端WEDL和C端WEII与LC3结合。

Objective:To determine the interaction between nuclear receptor co-activator 7(NCOA7)and microtubule-associated protein light chain 3(LC3),and identify the LC3-binding sites in NCOA7.Methods:The mass spectrometry which was performed in gastric cancer AGS cells and GST-LC3 pulldown assay which was performed in HEK293 cells were used for identification of the LC3 binding proteins,the molecular cloning method for construction of the truncation and point mutants of NCOA7;Western blotting was used to detect the expression level of NCOA7 truncatedΔN462,ΔN650,ΔN650-I749/750A proteins,and the GST-LC3 pulldown assay to identify the LC3-binding site in NCOA7.Results:The GST-LC3 pulldown assay combined with mass spectrometry identified NCOA7 as an LC3-binding protein.Western blotting results showed that different truncations and point mutants of NCOA7 were expressed.GST-LC3 pulldown NCOA7 mutants assay showed that the N-terminal WEDL motif and C-terminal WEII motif of NCOA7 were LC3 binding sites.However,binding to LC3 of the WEII motif was inhibited at the 651~720 amino acid sequence of NCOA7,which is an adjustable LC3 binding site.Conclusion:NCOA7 is an LC3 binding protein.Its N-terminal WEDL and C-terminal WEII motifs bind to LC3.