AGPS在神经胶质瘤细胞中的相互作用蛋白及作用模式

Interactive protein and mode of AGPS in glioma cells

目的探讨胶质瘤细胞(U251细胞)中癌基因烷基甘油酮磷酸合酶(AGPS)的相互作用蛋白及作用模式。方法常规培养U251细胞并随机分为对照组、shR-AGPS-1组、shR-AGPS-2组,分别加入阴性对照慢病毒和不同沉默水平的AGPS shRNA慢病毒,用Western blotting法检测AGPS蛋白表达以验证沉默效率。通过免疫共沉淀技术和质谱技术鉴定AGPS相互作用的蛋白,用Western blotting法进行验证;用计算机模拟技术进行相互作用蛋白的同源模建并推测二者相互作用模式。结果与对照组比较,shR-AGPS-1组、shR-AGPS-2组AGPS表达低(P均<0.05),且shR-AGPS-1组AGPS表达低于shR-AGPS-2组(P<0.05)。将筛选获得的数据进行整理,初步判断不均一核糖核蛋白K(HNRNPK)为AGPS的靶蛋白。在AGPS抗体免疫共沉淀的细胞裂解物中可同时检测到AGPS蛋白、HNRNPK蛋白,表明AGPS与HNRNPK可形成复合体,二者均在细胞核中表达。计算机模拟结果显示AGPS和HNRNPK通过氨基酸残基以氢键和共轭、疏水键和静电力形式相互作用。结论胶质瘤细胞中HNRNPK是AGPS的相互作用蛋白,氢键和共轭、疏水键和静电力相互作用可能是其主要的作用模式。

Objective To explore the interactive protein and mode of oncogene alkylglycerone phosphate synthase(AGPS)in glioma U251 cells.Methods U251 cells were routinely cultured and divided into the control group,shRAGPS-1 group and shR-AGPS-2 group,respectively,which were added with the negative control lentivirus and AGPS shRNA lentiviruse with different silencing levels.Western blotting was used to detect the expression of AGPS protein to verify the silencing efficiency.The interactive protein of AGPS was identified by immunoprecipitation and mass spectrometry,and was identified by Western blotting.The homologous modeling of the interactive proteins of AGPS was established by computer simulation and the interaction mode was predicted.Results Compared with the control group,the expression of AGPS decreased in the shR-AGPS-1 group and shR-AGPS-2 group and the expression of AGPS was lower in the shRAGPS-1 than in the shR-AGPS-2 group(all P<0.05).It was predicted that heterogeneous nuclear ribonucleoprotein K(HNRNPK)was the target protein of AGPS,and both AGPS and HNRNPK were detected in the AGPS immunoprecipitation lysate,which indicated that AGPS and HNRNPK formed complexes,and both of them were expressed in the nucleus.The results of computer simulation showed that AGPS and HNRNPK were interacted through amino acid residues based on hydrogen bond,conjugation,hydrophobic bond and electrostatic force.Conclusion The HNRNPK is the interactive protein of AGPS in glioma,and the interaction of hydrogen bond,conjugation,hydrophobic bond and electrostatic force may be the main modes of action of AGPS.