候选蛋白多聚胞嘧啶结合蛋白1与泛素连接酶环指蛋白4交互作用的研究

Study on interaction of ring finger protein 4 with protein candidate poly(rC) binding protein 1

目的 :寻找与类泛素(small ubiquitin-like modifier,SUMO)介导的泛素连接酶(SUMO-targeted ubiquitin ligase,STUbL)环指蛋白(ring finger protein 4,RNF4)相互作用的蛋白以及底物,为相关疾病的分子靶向治疗提供理论依据和实验数据。方法:采用酵母双杂交技术在人脑互补DNA文库、高灵敏质谱分析技术在RNF4过表达细胞系中筛选出一批与RNF4相互作用的候选蛋白,通过免疫共沉淀(CO-IP)实验验证候选蛋白是否确实与RNF4发生相互作用,采用相同实验检测候选蛋白是否可被SUMO化修饰。结果:酵母双杂交和高灵敏质谱分析技术筛选出77个候选蛋白。验证了采用2种方法均筛出的多聚胞嘧啶结合蛋白1[poly(rC)binding protein 1,PCBP1]可与RNF4交互作用,并且CO-IP实验证实PCBP1可以与SUMO相互作用。结论:PCBP1与RNF4存在交互作用,PCBP1蛋白可以被SUMO化修饰,RNF4可能通过调控SUMO化的PCBP1的降解来影响其功能。

Objective To find the proteins/substrates interacting with small ubiquitin-like modifier （SUMO）-targeted ubiquitin E3 ligase ring finger protein 4 （RNF4） and provide evidence of the molecular targets involved in the diseases. Methods Using yeast two-hybrid technology to screen human fetal brain library, as well as using high sensitive mass spectrometry technique to screen cells overexpressing RNF4 to identify candidate proteins interacting with RNF4. Coimmuno-precipitation（CO-IP） were performed to confirm the interaction between RNF4 and the candidate proteins, also to confirm the SUMOylation of candidate proteins Results About seventy-seven candidate proteins were found in yeast two- hybrid test and in mass spectrometry together. Poly（rC） binding protein 1 （PCBP1）, which was identified in both methods, was further studied. The interaction between RNF4 and PCBP1 was confirmed by CO-IP experiment, also the SUMOylation of PCBP1 was confirmed. Conclusions PCB151 is one of the candidate proteins interacting with RNF4. The sumoylation of PCBP1 was confirmed. RNF4 might regulate the function of PCBP1 through controlling its degradation.