与PIH1D1相互作用的蛋白分析

Analysis of the interaction proteins of PIH1D1

目的分析细胞中与PIH1D1相互作用的蛋白。方法构建稳定表达FLAG-HA双标签标记的PIH1D1蛋白的HEK293T细胞株,利用FLAG-HA串联亲和纯化(TAP)双标签纯化实验,对目的条带进行质谱分析。结果成功构建稳定表达FLAG-HA双标签标记的PIH1D1细胞株。通过质谱分析得到了PIH1D1相互作用的蛋白数据,包括细胞质内RNA PolⅡ组装复合物成员RPAP3、UXT、PFD2和PFD6等,凋亡复合物成员MONAD/WDR92等,钙调蛋白信号通路中的PIP和CALM1以及代谢通路中的PKM和LCN1等。结论 PIH1D1与细胞中RPAP3、UXT、PFD2、PFD6、MONAD/WDR92、PIP、CALM1、PKM和LCN1等相互作用,提示PIH1D1可能参与细胞中RNApolⅡ组装、细胞凋亡、钙调蛋白通路等多种生理过程。

Objective To investigate HEK293T cell strain was constructed the binding proteins of PIH1 D1. using a FLAG-HA double-tagged Methods A PIHID1 stable-expressed PIHIDI expressing plasmid. After per- forming the FLAG-HA tandem affinity purification （TAP） , the mass spectrometry was carried out to analyze the interaction protein complex of PIH1D. Results The FLAG-HA double-tagged PIHID stable-expressed HEK293T cell strain was obtained. We found a number of potential binding proteins of PIHID1 by means of the mass spectrometry, including RPAP3, UXT, PFD2 and PFD6 which were involved in RNAP ]I assemble factory and MONAD/WDR92 which was able to promote apoptosis. Besides we found PIP and CALM1 belong- ing to calmodulin-Ca2＋ complex, and PKM and LCN1 which were classic enzyme of metabolism as binding proteins of PIHID1 for the first time. Conclusions PIH1DI may participate in multiple biological processes including apoptosis, calmodulin-Ca（2 ＋ ） signaling pathway, RNA polymerase II assembling, and metabo- lism regulation through interaction with a RPAP3, UXT, PFD2, PFD6, MONAD/WDR92, PIP, CALM1, PKM and LCN1.