谭拥军 杨仕平 余雳 黄小芹 陈燕 谭桂湘

摘   要：为了鉴定转录因子FOXM1转录激活结构域（C-端第688位到748位氨基酸序列）的互作蛋白，以FOXM1的cDNA为模板，采用聚合酶链式反应（Polymerase Chain Reaction，PCR）方法扩增获得其转录激活结构域序列，克隆进入原核表达载体pGEX-4T2;利用大肠杆菌原核表达获得融合谷胱甘肽S-转移酶（Glutathione S-Transferase，GST）標签的重组蛋白GST-FOXM1688-748;通过GST-pulldown结合质谱方法鉴定FOXM1688-748的互作蛋白，成功构建了原核表达质粒pGEX-4T2-FOXM1688-748，获得了原核表达的重组蛋白GST-FOXM1688-748. 将质谱鉴定获得的互作蛋白进行分析和归类，预示一些互作蛋白可以参与激活FOXM1的转录活性，如RPN2、MISP、MCM7蛋白，一些互作蛋白可以参与调控FOXM1蛋白的稳定性，如USP9Y、CUL4A、HSPB1、BAG2蛋白. FOXM1蛋白的转录激活结构域与许多不同功能的蛋白发生相互作用，暗示该结构域具有重要的分子生物学作用，期望为以FOXM1为靶点的临床药物研发提供实验依据.

关键词：转录因子FOXM1;FOXM1688-748结构域;原核表达;相互作用

中图分类号：Q786                                   文献标志码：A

Identification of FOXM1688-748 Domain-interacting Proteins

TAN Yongjun†，YANG Shiping，YU Li，HUANG Xiaoqin，CHEN Yan，TAN Guixiang

（College of Biology，Hunan University，Changsha  410082，China）

Abstract：To identify the interacting proteins of transactivation domain of forkhead box protein M1 which is located on the 688th to the 748th amino acid sequence of C-terminal end，the cDNA of FOXM1 was used as a template， the transcription activation domain sequence was amplified by polymerase chain reaction and cloned into the prokaryotic expression vector pGEX-4T2. The recombinant protein GST-FOXM1688-748 fused with glutathione S- transferase tag was obtained by Escherichia coli prokaryotic expression. The interacting proteins were identified by GST-pulldown assay and mass spectrometry. The pGEX-4T2-FOXM1688-748 prokaryotic expression plasmid was constructed successfully and the FOXM1688-748 recombination protein was obtained. According to the mass spectrometry results， the interacting proteins were analyzed and classified to show that some of them activated the transcriptional activity of FOXM1， such as RPN2， MISP， and MCM7 proteins， and some of them regulated the stability of FOXM1 protein， such as USP9Y， CUL4A， HSPB1， and BAG2 proteins. The transcription activation domain of FOXM1 protein interacts with many proteins participating in different pathways， indicating that this domain has an important molecular biological role， which can expect to provide experimental basis for clinical drug development targeting FOXM1.

Key words：FOXM1;FOXM1688-748 domain;prokaryotic expression;interaction

FOXM1（Forkhead box M1）是转录因子FOX家族的成员，该家族成员都有一个保守的翼状螺旋DNA结合结构域[1]. FOXM1几乎在所有肿瘤细胞中高表达，是基因表达和细胞循环机制以及其他重要生理过程的关键调节器[2]，参与肿瘤细胞的增殖、迁移、侵袭、转移、上皮-间质转化、肿瘤血管生成、防止细胞过早衰老等过程[3-7]，它与癌症的发生和发展存在着至关重要的关系，已被确立用于癌症诊断、治疗和预后[8]. FOXM1蛋白主要包含3个结构域，分别是保守的DNA结合结构域（DBD）、N-端抑制结构域（NRD）、C-端转录激活结构域（TAD）[9]. FOXM1激活靶基因转录的机制中，通……