炎症应答中去泛素化酶圆柱瘤蛋白相互作用分子筛选和验证

Screening and validation of interacting molecules of deubiquitinase cylindromatosis in inflammatory responses

目的筛选炎症应答中与去泛素化酶圆柱瘤蛋白(CYLD)相互作用分子,探究并验证CYLD和参与NF-κB信号通路调节的桥梁蛋白14-3-3家族分子之间是否在细胞内存在相互作用。方法通过免疫共沉淀(Co-IP)飞行质谱筛选可能与CYLD相互作用分子,在不同的刺激条件下诱导骨髓原代巨噬细胞向M1和M2极化,利用qRT-PCR、Western blotting技术检测CYLD及可能相互作用的候选分子14-3-3在巨噬细胞内的表达水平。结果CYLD抗体可以捕获14-3-3蛋白全部的7种亚型(β、γ、ε、η、ζ、δ、θ),提示CYLD很可能与14-3-3蛋白存在相互作用,选取质谱结果中得到特异性肽段最多的3种14-3-3亚型γ、ε、η(14-3-3γ/ε/η),检测结果显示在M0、M1、M2型巨噬细胞中CYLD和14-3-3γ/ε/η均有表达。构建了Myc-CYLD和Flag-14-3-3γ/ε/η真核过表达载体,用Flag抗体成功沉淀出CYLD蛋白,Co-IP结果提示CYLD和14-3-3γ/ε/η三种亚型均存在相互作用。免疫荧光实验显示CYLD和14-3-3在巨噬细胞内存在共定位现象。结论巨噬细胞炎症应答中CYLD和14-3-3γ/ε/η三种亚型可能存在物理上的相互作用。

Objective To screen the molecules interacting with deubiquitinase cylindromatosis(CYLD)in inflammatory response,and to explore and verify the intracellular interaction between CYLD and bridge protein 14-3-3 family molecules involved in the regulation of NF-κB signaling pathway.Methods Molecules that might interact with CYLD were screened by co-immunoprecipitation(Co-IP)and flight mass spectrometry.Under different stimulation conditions,bone marrow primary macrophages were induced to polarize towards M1 and M2.qRT-PCR and Western blotting were used to detect the expression levels of CYLD and candidate molecule 14-3-3 that might interact with CYLD in macrophages.Results CYLD antibody could capture all seven subtypes of 14-3-3 protein(β,γ,ε,η,ζ,δ,andθ),suggesting that CYLD may interact with 14-3-3 protein.The three subtypes of 14-3-3(14-3-3γ/ε/η)with the most specific peptide segments were selected from the mass spectrometry.The results showed that CYLD and 14-3-3γ/ε/ηwere expressed in M0,M1 and M2 macrophages.Furthermore,Myc-CYLD and Flag-14-3-3γ/ε/ηeukaryotic overexpression vectors were constructed,and CYLD protein was precipitated by Flag antibody.Co-IP results indicated that CYLD could interact with 14-3-3γ/ε/η.Immunofluorescence assay showed that CYLD and 14-3-3 were co-localized in macrophages.Conclusion There may be physical interaction between CYLD and 14-3-3-γ/ε/ηin inflammatory response of macrophage.