The Investigation Of NEK7 Regulatory Signaling Protein With Mass Spectrometry

Inflammation is closely related to immunity and tumorigenesis.Inflammation can be induced by abnormal activation of the inflammasome.The most studied inflammasome is the NLRP3 inflammasome.It is interesting that it can recognize a variety of signal stimuli and ultimately activate NLRP3 through different mechanisms.More and more diseases are found to be closely related to NLRP3 inflammasome activation,such as gout,diabetes,atherosclerosis,Alzheimer’s disease,etc.Therefore,understanding the details of the assembly and activation of NLRP3 inflammasome and the development of molecular therapeutic drugs for its downstream moleculars will help us better understand the pathogenesis of inflammatory diseases,and provide a new theoretical basis for the treatment of NLRP3 inflammasome-related diseases in clinic.In 2016,a number of research groups identified a new component of NLRP3 inflammasome by different screen methods,NEK7,which played an important role in the activation of NLRP3 inflammasome by K+ outflow.Surprisingly,this protein is mainly involved in the formation of spindles and the separation of centrosomes in mitosis,and it has recently been discovered that it is also involved in the activation of NLRP3 inflammasome.So how does NEK7 respond to K+efflux and participate in the activation of NLRP3 inflammasome?Is there a signaling pathway that regulates the binding of NEK7 to NLRP3 to shift NEK7 from mitosis to inflammasome activation?To answer these questions,we first precipitated NEK7 and its interacting proteins by immunoprecipitation,then eluted with 3 X flag peptide,and carried out mass spectrometry amalysis after a series of treatments.We used bioinformatics methods such as gene ontology analysis,signal pathway analysis and protein-protein interaction analysis to analyze all the differential proteins in the experimental group,and then we consulted the relevant literature to find a protein which is most likely interacts with NEK7:RACK1(GNB2L1).We demonstrated the specific binding of NEK7 and RACK1 in HEK293T by CO-IP.Therefore,a novel NEK7 binding protein was identified by mass spectrometry.And we have reconstructed the functional NLRP3 inflammasome in HEK293T.We put the RACK1 protein into the signaling pathway of NLRP3 inflammasome activation to see the function role of RACK1,and further study the specific mechanism of NLRP3 inflammasome activation.