Mechanism Of B7-H4 Degradation By Ubiquitin Proteasome Pathway

Ubiquitin proteasome system is an extremely complex and highly specific protein degradation mechanism mediated by ubiquitin in eukaryotes,which can mediate the degradation of most proteins in cells.Many cellular processes such as cell growth and differentiation,regulation of cell cycle,DNA repair,immune response and apoptosis are closely related to the regulation of ubiquitination modification.The dysfunction of ubiquitin proteasome system can cause many serious diseases in human beings,and the abnormality of some key enzymes is closely related to the formation and development of tumors.In the previous studies related to B7-H4 molecules,we found that B7-H4 was significantly up-regulated by Westernblot detection after cytokine EGF stimulation or expression of EGF receptor(EGFR),but the transcriptional level did not show the same trend.In addition,related studies have found that the downstream proteome regulated by cytokine EGF contains a large number of signal proteins,ubiquitin and deubiquitinase,transport proteins and transcriptional translation related proteins,etc.,coupled with the evidence of previous immunoprecipitation results in our laboratory,there are indeed many ubiquitin-proteasome-related molecules:in B7-H4 interaction proteins.This suggests that the expression of B7-H4 in cells may also be regulated by this system.On this basis,we found that there is ubiquitin regulation in B7-H4 in vivo.In the case of EGF stimulation or expression of EGFR mutants at the cellular level,the changes of ubiquitin levels on B7-H4 molecules were detected by means of co-immunoprecipitation and immune hybridization,indicating that EGF pathway can indeed change its ubiquitin level and then its protein level.In addition,we used the B7-H4-3XFlag plasmid constructed in our laboratory and 8 human-encoded ubiquitin-specific protease plasmids for co-transfection,and then found that USP2 had a significant effect on the ubiquitin level of B7-H4 by co-immunoprecipitation.On this basis,we found that there is a certain interaction between USP2 and B7-H4 in vivo by overexpression combined with immunoprecipitation.This thesis mainly includes the following two parts:Part ? EGFR mutants increase the protein level of B7-H4 by enhancing the stability of B7-H4Objective:To investigate whether there is ubiquitin regulation in B7-H4 and whether there is ubiquitin-related regulation in the regulation of post-translational modification of B7-H4 by EGF pathway.Methods:Ubiquitin proteasome inhibitor MG132 was used to treat H23 and A549 cells to detect the protein expression of B7-H4.The eukaryotic expression vector of B7-H4 labeled with Flag was constructed,and EGFR mutant,B7-H4 and ubiquitin plasmids were transfected into 293T cells by polyethyleneimine(PEI).The total protein was extracted at 24 h.B7-H4 was enriched by immunoprecipitation to detect the level of ubiquitin on the molecule.Results:The B7-H4 plasmid labeled with Flag was successfully constructed,B7-H4 in many kinds of cells was up-regulated in varying degrees under the treat of MG 132,and the ubiquitin level of B7-H4 was down-regulated after co-transfection of EGFR mutant and B7-H4 plasmids for immunoprecipitation.Conclusion:It is confirmed that B7-H4 is indeed regulated by ubiquitin system in vivo,and the upregulation of B7-H4 by EGFR mutants may be achieved by changing the level of ubiquitin modification of B7-H4 to improve its stability.Part ? The regulation of deubiquitin enzyme USP on B7-H4 and identification of its interactionObjective:To find the related deubiquitin enzyme DUB,which can specifically remove ubiquitin from B7-H4 molecule and verify the interaction between the two molecules in vivo.Methods:The expression of endogenous and exogenous B7-H4 was detected by transfection of related deubiquitination enzyme plasmids in cells,and the possible interaction between DUB,and B7-H4 was preliminarily screened.The effect of corresponding DUB on the level of ubiquitin in B7-H4 was detected by immunoprecipitation.Finally,forward and reverse immunoprecipitation were used to identify the interaction between the two proteins.Results:The results of Western blot and co-immumoprecipitation showed that overexpression of USP2 in several kinds of DUBs could significantly reduce the level of ubiquitin on B7-H4 molecule,and results of forward and reverse immunoprecipitation also showed that the two proteins interacted in vivo.Conclusion:USP2 can regulate the level of ubiquitin on B7-H4.USP2 may be a DUB that can specifically regulate B7-H4 protein...