The Study Of Interacting-protein Of Deubiquitase CYLD And Its Function On Regulating Macrophage Activation

?Backgroud?Macrophage is a kind of immune cell with high functional plasticity,which is ubiquitous in mammalian tissues and organs.Macrophages in physiological state participate in the maintenance of tissue homeostasis and integrity by identifying and engulfing exogenous pathogens.However,in pathological state,the function of macrophage is more complicated,and plays a double sword role.Under LPS and IFN-? stimulation,macrophage can polarize to M1 macrophageswhich exhibits the phenotype of pro-inflammation,anti-tumor and tissue destruction;while macrophages induced by IL-4 and IL-13 undergo M2 polarization and possess the phenotype of anti-inflammation,protumor and tissue remodeling.For example,in the progression of liver fibrosis,macrophages can secret proinflammatory cytokines to promote inflammationand liver fibrogenesis,and on another hand,it can participate in the extracellular matrix degragation,leading to tissue repair and remodeling.In order to investigate the function of macrophages in liver fibrosis,our team's previous study found that specifically knockout Notch signal in macrophages could upregulate the expression of CYLD which can inhibite the activation of NF-?B,consequently,to alleviate liver fibrosis by preventingactivation of hepatic stellate cells.However,the detailed molecular mechanisms of CYLD regulating macrophages activation remain unclear.Cyld is a tumor suppressor gene,and its mutation causes the occurrence of cylindroma.In addition,CYLD is also a kind of deubiquitase,which can specifically remove the K63-linked polyubiquitin chains of target proteins in order to alter their function.NF-?B and JNK signaling are both mediated by CYLD via its specific TRAF2 and NEMO binding sites on protein sequence.In order to find substrates for CYLD,we used immunoprecipitation combined with flight mass spectrometry to obtain a series of potentially interacting molecules.14-3-3 protein as a scaffold protein which was involved in the regulation of the NF-?B signaling pathway has attracted our attention.Then,we hadinvestigated the interaction between CYLD and 14-3-3 through varios molecular biology techniques,and explored its role and mechanism on regulating macrophage survival and activation.?Aims?1.To screen proteins that can interact with CYLD.2.To confirm the interaction between CYLD and 14-3-3.3.To explore the key phosphorylation interaction sites between CYLD and 14-3-3.4.To investigate the effects of 14-3-3 orCYLD when they can interact with each other.5.To understand the function of interaction between CYLD and 14-3-3 on macrophage activation and survival.?Methods?1.Search the potential proteins that can interact with CYLD by immunoprecipitationflight mass spectrometer technique.2.Culture Bone marrow derived-macrophages,and polarize them with different cytokine.qRT-PCR and Western blot technique are perfomed to detect the expression of CYLD and 14-3-3 in different polarized macrophages.3.Confirm the interaction between CYLD and 14-3-3 by Co-immunoprecipitation,mammalian two-hybrid and GST-pull-down assays.4.Mutate cyld gene by overlap extension PCR,and investigate the interaction between mutant CYLD and 14-3-3 via co-immunoprecipitation assay.5.Construct eukaryotic expression vector of HA-IKK?,and explore whether HA-IKK? can affect the interaction between CYLD and 14-3-3.6.Detect the total protein level of CYLD in macrophages after stimulating with LPS,CHX,MG-132,BV02 and zVAD-fmk.7.Explore the effect of 14-3-3 on the subcellular localization of CYLD by Western blot.8.Detect the ubiquitination of 14-3-3 by immunoprecipitation.9.Investigate the impact of BV02 on macrophage polarization with qRT-PCR.10.Construct the lentiviralvectorof cyld overexpression and knockdown using shRNA,and infect Raw264.7 cell,and establishstablecyld overexpression and knowdown cell line with Puromycin dihydrochloride selection.?Results?1.14-3-3 protein(?????),as a potential interacting molecules of CYLD,was captured using immunoprecipitationand flight mass spectrometer2.Successfully gained different polarization(M0,M1,M2)of bone marrow derivedmacrophages with M-CSF and other cytokine stimulation.qRT-PCR showed that cyld and 14-3-3 genes expressed differently in each phenotype of macrophages.cyld and 14-3-3? expression level increased in M1 macrophage,14-3-3? expression level increased in M2 macrophage,and 14-3-3? expression level increased in M2 macrophage and decreased in M1 macrophage,suggesting that CYLD and 14-3-3 may play a role in macrophage polarization.3.CYLD could interact with 14-3-3? ? ? and ? using co-immunoprecipitation.However,their interaction showed negative using mammalian two-hybrid and GST-pulldown experiments,indicating that their interaction might strictly depend on their spaceconformation and post-translational modifications.4.Successfully constructed mutant CYLD eukaryotic expression vectors,and Coimmunoprecipitation assay showed that phosphorylated S414 site of CYLD wasresponsibe for interaction between CYLD and 14-3-3.However,their interactioncould not be enhanced understimulation with HA-IKK?(IKK? can phosphorylate CYLD S414)kinase.5.Under the stimulation of LPS,CYLD in macrophages would be cleaved into 25 kD N-terminal and 80 kD C-terminal fragments by caspase8,and then the 80 kD C-terminal fragment was degraded by proteasome.However,the cleaved procedure does not require 14-3-3.Pan-14-3-3 cannot regulate the subcellular localization of CYLD,and macrophage polarization does not depend on pan-14-3-3.6.14-3-3(?????)cannot be modified by ubiquitination.Thus,they are not the substrates of the deubiquitination CYLD.7.Successfully constructed the lentiviral vectorofcyld overexpression and Knockdown using shRNA,and stable transfectionRaw 264.7 cell line was established,which can be used further to investigate the function of CYLD in macrophage.?Conclusions?1.CYLD and 14-3-3 can interact with each other,which mainly relys on the phosphorylation of S414 siteof CYLD.2.UnderLPS stimulation,the members of 14-3-3 family do not take part in the cleavage of CYLD by caspase8.14-3-3 molecules are not the substrates of the deubiquitination CYLD.Pan 14-3-3 does not influence the nucleic location of CYLD after LPS stimulation.And Pan 14-3-3 does not regulate the macrophage polarization.3.We constructed the lentiviral vector of cyld overexpression and knockdown,and stable transfection Raw 264.7 cell linewas establishedafter infecting with cyld overexpression and knockdown vector,which can be further usded for investigating the function of CYLD in macrophage survival and function.In summary,through a series of protein interaction assays,we found that the deubiquitase CYLD can interact with somemoleculesof 14-3-3 family.However,14-3-3 is not an ubiquitylation substrate for CYLD.Their interaction does not affect the macrophage polarization.We further constructed a lentiviral vector for over-expression and knockdown of CYLD,which setup the foundation for the further study of the molecularmechanism of CYLD on regulating macrophage survival and activation.