Mdfic与Rhox5蛋白相互作用的关键结构域鉴定  

Identification of the Critical Structures for Mdfic-Rhox5 Interaction

作　　者：郭芬[1] 欧淑芳[2] 初彦辉[3] 李月琴[2] 王丁丁[1] 周天鸿[2] 

机构地区：[1]广东药学院生命科学与生物制药学院,广州510006 [2]暨南大学基因工程药物国家工程研究中心,广州510632 [3]牡丹江医学院,黑龙江牡丹江157011

出　　处：《中国生物化学与分子生物学报》2012年第10期919-925,共7页Chinese Journal of Biochemistry and Molecular Biology

基　　金：广东省教育厅高水平科研项目(自然科学,No.LYM10094);广东省优秀博士论文作者资助项目(No.sybzzxm201051);高等学校博士学科点专项科研基金(No.20060559006);广东药学院科研启动基金项目(No.2007SMK02)~~

摘　　要：Mdfic(MyoD family inhibitor domain containing)是一个新发现的含有MyoD抑制素结构域(I-mfa domain)的转录调控因子,可能在肌细胞的分化过程中发挥重要作用.小鼠Rhox5为同源异型框基因,隶属于Rhox基因簇(reproductive homeobox on the X chromosome genes cluster)β亚簇.在前期证实Mdifc能结合Rhox5蛋白的基础上,进一步鉴定两者相互作用的关键结构域.生物信息学分析Mdfic的氨基酸序列,PCR方法扩增Mdfic A截短型片段(第72～247位氨基酸残基),含保守的I-mfa结构域;双向酵母双杂交和体外GST-Pull down结果表明,该截短型片段可以与Rhox5蛋白结合,且结合力度较完整的Mdfic蛋白强;将Mdfic A片段划分为两段:Mdfic B(72～191 aa,不含I-mfa结构域)和Mdfic C(191～247 aa,含I-mfa结构域).结果表明,含保守I-mfa结构域的Mdfic C截短型片段丧失了与Rhox5蛋白结合的能力,而不含I-mfa结构域的Mdfic B截短型片段可以结合Rhox5蛋白.鉴于Mdfic蛋白的非I-mfa结构域在Rhox5/Mdfic结合中发挥关键作用,Rhox5与Mdfic的结合可能进一步调控由Mdfic的I-mfa结构域参与的其他转录因子(如MyoD)的调控,三者形成一个复杂的调控网络,共同参与肌细胞发生及分化的调控.Mouse Mdfic （MyoD family inhibitor domain containing） protein contains an I-mfa domain. Mdfic is a newly discovered transcriptional factor that may play important roles in myogenesis. Mouse Rhox5 gene is a homeobox gene which belongs to the 13 subcluster of Rhox cluster （reproductive homeobox on the X chromosome）. In our previous study, Mdfic was found to interact with Rhox5 both in vitro and in vivo. In this study we investigated the critical structure involved in Mdfic/Rhox5 interaction. Mdfic A truncated fragment （from 72 aa to 247aa） , including the I-mfa domain, was obtained. Two-way yeast two-hybrid assay and GST-pull down assay indicated that Mdfic A truncated fragment displays a little higher affinity with Rhox5, compared to Mdfic protein. Furthermore, Mdfic A truncated fragment was divided into two parts： Mdfic B （from 72 aa-191aa, without I-mfa domain） and Mdfic C（from 192 aa-247aa, including I-mfa domain）. Surprisingly, the Mdfic C truncated fragment lost the ability to interact with Rhox5, while the Mdfic B truncated fragment bound to Rhox5 protein. These indicated that the critical structure of Mdfic/Rhox5 was 192 aa-247 aa, not the I-mfa domain, which suggested that Mdfic/Rhox5 interaction might regulate the event controlled by Mdfic and other transcriptional factors. Mdfic/Rhox5/transcriptional factor may form a complex regulatory net to regulate the determination and differentiation events during myogenesis.

关 键 词：转录因子 Mdfic Rhox5 相互作用关键结构 肌细胞发生与分化 

分 类 号：Q51[生物学—生物化学]