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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 search for proteins and substrates that interact with SUMO-targeted ubiquitin-ligase (STUbL) ring finger protein 4 (RNF4) , Provide theoretical basis and experimental data for molecular targeted therapy of related diseases. Methods: A series of candidate proteins interacting with RNF4 in RNF4 overexpression cell line were screened by yeast two-hybrid technique in human brain complementary DNA library and highly sensitive mass spectrometry. Candidate was confirmed by co-immunoprecipitation (CO-IP) Whether the protein actually interacts with RNF4 and whether the candidate protein can be modified by SUMO using the same experiment. RESULTS: Totally 77 candidate proteins were screened by yeast two-hybrid assay and high sensitive mass spectrometry. It was confirmed that poly (cC) binding protein 1 [PCBP1] screened by two different methods could interact with RNF4, and COPIP confirmed that PCBP1 could interact with SUMO. CONCLUSION: PCBP1 interacts with RNF4. PCBP1 protein may be modified by SUMO. RNF4 may affect its function by regulating the degradation of SUMO-treated PCBP1.