USP12 Regulates IFNs-mediated Signaling Pathway And Antiviral Activity

Objective:Interferons(IFNs)are very important cytokines in both innate immunity and antiviral immunity.IFNs-mediated antiviral immunity plays a critical role in host resistance to viral infection.Recent studies have mainly focused on the regulation of phosphorylation of IFNs signaling proteins.With the in-depth study of protein ubiquitination modifications,more and more attention has been paid to the ubiquitination and deubiquitination of proteins.Our previous studies found that the deubiquitinase USP2a significantly enhanced IFNs signaling pathway.In addition,we found that another deubiquitinase USP12 was also involved in the regulation of IFNs signaling.Interestingly,we noticed that this effect of USP12 does not depend on its deubiquitinase activity.Further studies suggest that USP12 could play a role in regulating IFNs signaling by affecting CBP.Therefore,this study aims to explore how USP12 affects the key protein CBP and subsequently regulates IFNs signaling pathway and IFNs antiviral function.This study could provide potential therapeutic strategies for enhancing the antiviral efficacy of IFNs.Methods:(1)Cells were transfected with Flag-HA-USP12(FH-USP12)plasmids.The whole cell lysates(WCL)were subjected to immunoprecipitation using Flag(M2)beads.We performed mass spectrometry analyses to identify the binding proteins of USP12.We identified CBP that could interact with FH-USP12.Next,we explored which domain of CBP could interact with USP12.And then we analyzed whether USP12 regulates CBP protein levels.(2)Cells were transfected with USP12 knockdown plasmids.Then we analyzed whether USP12 could affect the acetylation of IFNAR2 and STAT2 induced by CBP in IFNs signaling pathway by Western Blot.And then we further explored whether USP12 regulates the formation of ISGF3 trimer complex.(3)Western Blot and Confocal microscopy assay were used to analyze the intracellular localization of USP12 under IFNs stimulation.(4)Cells were transfected with FH-USP12 or shUSP12 plasmids.The effects of USP12 on the acetylation of STAT1 and p-STAT1 were analyzed by Western Blot after nuclear separation.In vitro acetylation assay was performed to analyze whether USP12 regulates STAT1 acetylation.The levels of p-STAT1 in nucleus were also analyzed.(5)Cells were transfected with FH-USP12 or shUSP12 plasmids.With IFNs stimulation,p-STAT1 levels were detected by Western Blot.ISRE-luciferase activity was also analyzed by the Dual luciferase reporter gene technique.Representative ISGs mRNA levels were measured by RT-qPCR.In the end,we sought to determine whether USP12 regulates IFNs-mediated antiviral response by observing VSV-GFP fluorescence intensity.(6)We analyzed whether USP12 regulates IFNs signaling through its deubiquitinase activity by Western Blot,Dual luciferase reporter gene technique,RT-qPCR.(7)USP12-KO cell lines were made by the CRISPR/Cas9 technique.The effects of USP12 on IFNs signaling pathway were analyzed by Western Blot,Dual luciferase reporter gene technique,RT-qPCR.Results:(1)Mass spectrometry results showed that USP12 could interact with the acetyltransferase CBP;USP12 did not affect the protein level of CBP but affected the protein acetylation mediated by CBP by binding to the HAT region of CBP.(2)USP12 inhibited the regulation of CBP on the acetylation of IFNAR2 and STAT2 in IFNs signaling pathway.Further studies have found that USP12 reduces the formation of ISGF3 trimer complex by affecting the acetyltransferase activity of CBP.(3)Under IFNs induction,USP12 translocated from the cytoplasm to the nucleus.(4)USP12 decreased the acetylation of p-STAT1 in the nucleus.After knocking down USP12,the acetylation of p-STAT1 increased significantly.Further studies showed that USP12 could decrease the acetylation of p-STAT1,inhibit the interaction between p-STAT1 and TCPTP,and up-regulate the protein level of p-STAT1 in the nucleus.(5)USP12 significantly increased the total p-STAT1 levels.USP12 significantly increased IFNs-induced ISRE transcriptional activity by the double luciferase reporter gene technology.USP12 significantly enhanced IFNs-induced ISGs-mRNA levels by RT-qPCR.VSV-GFP fluorescence intensity was observed by immunofluorescence,and the results showed that USP12 enhanced IFNs-mediated antiviral function.(6)USP12 regulated IFNs signaling pathway and IFNs antiviral function independently of its deubiquitinase activity.(7)USP12-KO cell lines were made by the CRISPR/Cas9 technique.After knocking out USP12,IFNs signaling pathway was inhibited and IFNs-mediated antiviral immune response was reduced.Conclusions:USP12 inhibits CBP-mediated acetylation by interacting with the HAT region of CBP,thereby inhibiting the interaction between p-STAT1 and the phosphatase TCPTP in the nucleus and subsequently stabilizing the levels of p-STAT1 in the nucleus.These effects of USP12 positively regulate IFNs signaling pathway and ultimately enhance the antiviral function of the host.