Effects And Mechanisms Of The E3 Ubiquitin Ligase HERC6 Regularing ERK Signaling Pathway In Dendritic Cell Regulation

mHERC6 is an E3 ubiquitin ligase,which destroys virus replication through ISGylation protein.However,the exact role of mHERC6 in dendritic cells has not been determined,and the functions and molecular mechanisms of dendritic cell activation and immunogenicity regulation in immune diseases mediated by mHERC6 are still unclear.In this study,we found that mHERC6 plays a crucial role in inhibiting the inflammatory immune response.In addition,mHERC6 also inhibits the antiviral effect in vivo.Further studies showed that mHERC6 inhibited the production of inflammatory cytokines in dendritic cells by regulating ERK signaling pathway.Finally,we demonstrated that mHERC6 degrades B-Raf and C-Raf through proteasome pathway,thereby activating ERK signaling pathway.In conclusion,the results of this study reveal that mHERC6 targets B-Raf and C-Raf and plays an important role in regulating dendritic cell function.It is suggested that mHERC6 may serve as a potential target in regulating dendritic cell function.Methods1.After Herc6^fl/fl mice and Herc6^fl/fl;CD11c-Cre mice were infected with HSV,the spleens and lungs were ground into homogenate on the seventh day,and dendritic cells were isolated from single cells of the spleen.Half of the cells were detected by real-time quantitative PCR（qRT-PCR）to detect their mRNA,the rest of the cells were checkeded by the flow cytometry.At the same time,the eye blood and the secretion of inflammatory cytokines of the supernatant of spleen and lung homogenate were detected by enzyme-linked immunosorbent assay（ELISA）;2.Lipopolysaccharide（LPS）stimulated murine bone marrow derived dendritic cells（mBMDCs）of Herc6^fl/fl and Herc6^fl/fl;CD11c-Cre mice.The mRNA expression of cytokines was detected by qRT-PCR,the release of cell factors ware checked by ELISA,the amount of costimulatory molecules were checked by the flow cytometry;3.LPS stimulated mBMDCs from Herc6^fl/fl;CD11c-Cre mice and their control groups,co cultured with CD4⁺ T cells,and flow cytometry was employed to check the specific factors of CD4⁺T cells and the expression of intracellular cytokines;4.LPS stimulated mBMDCs from Herc6^fl/fl;CD11c-Cre mice and their control groups,and Western Blot（WB）was used to detect the changes of signal pathway molecules at different times of stimulation;5.Through pharmacological specific inhibition of mBMDCs related proteins from Herc6^fl/fl;CD11c-Cre mice and their control group,WB was employed to check expression of related proteins at different times when LPS stimulated,qRT-PCR was employed to check mRNA expression of cytokines.6.LPS stimulated mBMDCs from C57BL/6 wild-type mice,and co-immunoprecipitation（COIP）was used to detect the interaction between mHERC6 and B-Raf,C-Raf;7.Flag-mHERC6 and Myc-B-Raf or Myc-C-Raf were co-transfected into 293T cells,and the interaction between mHERC6 and B-Raf,C-Raf was detected by COIP;8.Flag-HERC5,V5-Ub and Myc-B-Raf or Myc C-Raf were co-transfected into 293T cells.COIP was used to check the impact of HERC5 on the ubiquitination of B-Raf and C-Raf.Results1.mHERC6 inhibits immune response in dendritic cells;2.mHERC6 inhibits the immune response of mice;3.The antiviral effect of mHERC6 in mice;4.mHERC6 activates ERK signaling pathway in dendritic cells;5.mHERC6 targets B-Raf and C-Raf and degrades them through proteasome;6.HERC5 makes B-Raf and C-Raf ubiquitin to degrade them.ConclusionmHERC6 targets B-Raf and C-Raf in dendritic cells,regulates ERK signaling pathway and inhibits the expression of IL-1β、IL-6、IL-12、TNF-α.Also mHERC6 inhibits its regulation of Th1 and Th17 cells differentiation,thereby inhibiting the antiviral effect in vivo,but at the same time inhibiting the "cytokine storm" caused by viral infection in mice to improve the survival rate of mice.