Identification Of MeCP2 Interacting Protein And Investigation Of The Relationship Between MeCP2 And CK2

Research background and Objective:Methylated-CpG-binding protein 2(MeCP2)was first identified as a protein that specifically binds to methylated DNA,and its expression in the brain is significantly higher than that of other tissues,mainly distributed in the nucleus.With the deepening of scientific research,it is now believed that MeCP2 has multifunctional regulatory gene expression(which can be used as a transcriptional repressor and activator)and epigenetic regulation-related functions.MeCP2 interacts with various proteins to form protein complexes through different domains,and plays an important functional role in biological processes such as neural development,cell differentiation,cell cycle and apoptosis.When MeCP2 is abnormal,it will cause a variety of nervous system related diseases,such as Rett syndrome,which is a typical neurodegenerative disease caused by mutations in the MeCP2 gene.However,at present,the functional role of MeCP2 in the nervous system and the pathological mechanism of the occurrence of related diseases are not yet clear.Therefore,this experiment aims to explore the MeCP2 interacting proteins in brain tissue and the functional relationship between these proteins to provide some clues and support for the further study of MeCP2 protein function.Methods:Firstly,the protein complexes of MeCP2 in different brain tissues were identified by the method of coprecipitation protein mass spectrometry(coip-ms),and the biological processes and functions involved in the interaction proteins with high reliability were analyzed by the method of bioinformatics.CoIP-WB experimen was used to verify the in vivo interaction.The fusion protein was obtained by prokaryotic expression and purification technology,and the surface plasmon resonance experiment and GST pull-down experiment were carried out to verify the interaction in vitro.Finally,the interaction between MeCP2 and CK2 kinase was confirmed by Western blot and protein mass spectrometry.Results:The results of CoIP-MS experiments and related bioinformatics analysis showed that the identified MeCP2 interacting proteins and cell adhesion,RNA splicing,synaptic transmission,cell differentiation,subcellular localization,mRNA metabolism regulation,apoptosis,mitosis,Protein folding modification and other biological processes.CoIP-MS experiment results and CoIP-WB verification found that MeCP2 interacts with CK2 kinase in vivo,forming a protein complex in the cell.The results of GST pull-down experiments show that both catalytic subunits of CK2 kinase can directly interact with MeCP2 in vitro.Finally,in the in vitro phosphorylation experiment,we found that CK2 kinase can phosphorylate the MeCP2 protein in vitro.After identification of the modified sites in the protein profile,we found that the phosphorylation sites are shown in Ser13 and Ser204.Conclusion:MeCP2 may participate in a variety of biological processes in different forms of complexes in the nervous system,and play an important role in epigenetic regulation.In vitro and in vivo experiments confirmed that MeCP2 directly interacts with CK2 kinase.After functional verification,we found that CK2 can phosphorylate the two sites of MeCP2 Ser13 and Ser204 in vitro,providing direct evidence for the presence of two phosphorylable sites Serl3 and Ser204 and their upstream phosphokinases in MeCP2.