HepG2细胞中RORγ相互作用蛋白的筛选及鉴定

Identification of interacting proteins of retinoid-related orphan nuclear receptor gamma in HepG2 cells

目的筛选HepG2细胞中转录因子RORγ(the retinoid-related orphan nuclear receptor gamma)相互作用蛋白,并对这些蛋白进行初步鉴定,为阐述RORγ介导调节HepG2细胞中各种生理学进程提供理论依据。方法从HepG2细胞中克隆RORγ基因并连接入载体pCeMM CTAP(SG)中形成RORγ-CTAP(SG)融合基因,再将其亚克隆入含嘌呤霉素抗性基因的载体质粒中,构建pMSCVpuro RORγ-CTAP(SG)质粒;稳定转染HepG2细胞并对RORγ-CTAP(SG)融合基因的表达和定位进行检测后,进行大规模扩增培养;用TAP(串联亲和纯化)方法捕获RORγ相互作用蛋白,通过银染找出差异性蛋白条带,质谱鉴定得到候选RORγ相互作用蛋白;用免疫共沉淀方法对候选蛋白RORγ相互作用蛋白进行验证鉴定。结果成功构建了质粒pMSCVpuro RORγ-CTAP(SG)并得到稳定转染HepG2细胞株,同时RORγ-CTAP(SG)融合基因能定位表达于细胞核内;通过TAP方法获得了RORγ蛋白复合物,然后通过串联质谱分析和数据库搜索从银染差异性显著蛋白条带中找到7个候选RORγ相互作用蛋白;用RORγ蛋白进行免疫共沉淀,对纯化出的7个RORγ相互作用蛋白分别检测,证实了RIP140,HSP90和RORγ在HepG2中有相互作用关系。结论筛选并鉴定了HepG2细胞中蛋白RORγ的相互作用蛋白RIP140和HSP90,这些研究发现支持了RORγ是共调解蛋白依赖的转录因子且以复合物形式行使功能的假说。其中,HSP90可能扮演分子伴侣角色,而RIP140在HepG2细胞中则可能充当RORγ蛋白的转录调控伙伴蛋白,具体机制有待进一步研究。

Capturing proteins interacting with the transcriptional factor RORγ（the retinoid-related orphan nuclear receptor gamma） in HepG2 cells,and subsequently identifying the candidate proteins,to provide theoretical prove for RORγ-mediated regulatory processes of HepG2 cells.RORγ was cloned from HepG2 cells and integrated into pCeMM CTAP（SG） vector to get the fusion gene RORγ-CTAP（SG）,which was then subcloned into the pMSCVpuro vector to obtain the puromycin-resistant recombinant plasmid pMSCVpuro RORγ-CTAP（SG）.After the plasmids pMSCVpuro RORγ-CTAP（SG） and pMSCVpuro CTAP（SG） were stablely transfected into HepG2 cells,Western blot assay and Immunofluorescence staining were used to determine whether RORγ were expressed correctly in HepG2 cells.The stablely transfected cells were cultured in large scale,TAP（tandem affinity purification） method,MS（mass spectrometry） and coimmunoprecipitation were used to capture and identify RORγ-interacting proteins.The results displayed that the construct of pMSCVpuro RORγ-CTAP（SG） and nuclear localization of RORγ-CTAP（SG） fusion gene in stably-transfected HepG2 cells were as expected;isolation and identification of RORγ protein complexes by TAP/MS strategy outcame seven candidate RORγ interacting proteins;coimmunoprecipitation of the RORγ complex confirmed the interaction among RIP140,HSP90 and RORγ in the HepG2 cells.In conclusion,we have identified and verified RIP140 and HSP90 as the interactors of RORγ in a complex format in HepG2 cells.These findings support the hypothesis that RORγ is a coregulator-dependent transcription factor and functions in a complex.HSP90 may act as a chaperone by binding to RORγ protein and helping RORγ execute its regulatory roles involved in liver metabolism and other biological events while RIP140 might function as a coregulatory partner of RORγ in HepG2 cells,which has to be clarified in the future.