GSTpull-down联合质谱鉴定BAG结构域相互作用蛋白

Screening Interacting Proteins for BAG Domains of BAG Family Proteins by GST pull-down Coupled with LC-MS/MS

目的:BAG结构域(BAG domain,BD)为BAG家族蛋白的基本功能结构域,通过对BAG家族蛋白6个成员的9个BDs的相互作用蛋白进行分析,以探明不同BD相互作用蛋白的异同点并为研究BAG家族蛋白多样性生物功能的分子机制提供理论依据。方法:构建p-GEX-4T2-BDs重组子并转化E.coli BL21(DE3)经IPTG诱导表达GST-BDs融合蛋白并纯化。采用GST pulldown技术联合高效液相色谱串联质谱(LC-MS/MS)的策略对BDs相互作用蛋白进行定性定量分析。最后,用DAVID(The Database for Annotation,Visualization and Intergrated Discovery)和cytoscape对BDs相互作用蛋白进行GO(Gene Ontology)功能分析及KEGG(Kyoto Enyoolpedia of Genes and Genomes)通路分析。结果:在Hela细胞的胞浆蛋白中总共鉴定到370个潜在的BDs相互作用蛋白,主要为核糖体蛋白(ribosomal proteins)、翻译起始因子(Eukaryotic translation initiation factors)、翻译延长因子(Eukaryotic translation elongation factors)、泛素化-蛋白酶体相关蛋白(ubiquitin-proteasome associated proteins)及HSP40家族蛋白。GO功能富集分析结果显示,BDs相互作用蛋白涉及多种生物学功能,包括细胞内蛋白质质量控制(protein quality control)、糖代谢(glycolysis)、免疫调控(immune response)、应激反应(stress response)、细胞周期(cell cycle)等。KEGG通路分析结果表明BDs相互作用蛋白参与多条细胞内重要的信号通路,包括FGF信号通路(FGF signaling pathway)、EGF受体信号通路(EGF receptor signaling pathway)、PDGF信号通路(PDGF signaling pathway)、Ras通路(Ras pathway)等。结论:BAG家族蛋白不同成员的BD所介导的蛋白-蛋白相互作用既有共性又有特异性,BAG家族蛋白通过BDs介导多种蛋白相互作用并参与细胞内多条重要的信号通路来调控细胞内蛋白质稳态、糖代谢、免疫反应、应激反应、细胞周期等过程。

The Bcl-2-associated athanogene （BAG） family proteins are BAG-domain （BD） containing protein and are evolutionally conserved among eukaryotes. BAG family proteins play a critical role in a wide variety of cellular processes, such as cell survival, proliferation, migration, apoptosis, stress response and neural differentiation. BDs are responsible in binding HSP70 to BAG proteins. However, how BDs mediate the protein complexes assembly to govern the diverse functions of BAG proteins is needed further studies. To explore the roles of BDs in BAG family proteins, GST-BDs pull-down coupled with LC-MS/MS were performed to screen the BDs associated proteins in cytosolic extracts from Hela cells. 370 potential BDs interacting proteins were identified. GO （Gene Ontology） annotation analysis shows that BDs interacting proteins function in cellular protein quality control, glycolysis, immune response, stress response, ubiquitin-proteasome regulation, cell cycle process. In particular, KEGG （Kyoto Enyoolpedia of genes and Genomes） pathway enrichment analysis reveals that BDs interacting proteins involve in several important pathways, including FGF signaling pathway, EGF receptor signaling pathway, PDGF signaling pathway and Ras pathway. In addition,different BDs can specifically binding different partners. Overall, this expands knowledge of the BDs interactome, provides a valuable resource for understanding the roles of BDs in mediating BAG proteins＇ different cellular functions.