STAT1 Linear Ubiquitination-mediated Regulation Of IFN Antiviral Signaling Pathway

Objective:Protein ubiquitination modification is one of the key regulatory mechanisms controlling multiple intracellular signaling pathways.In addition to the seven types of ubiquitination modifications(K6,K11,K27,K29,K33,K48 and K63),the newly identified linear ubiquitination modification has attracted much attention in recent years.At present,linear ubiquitination has been recognized to play an important role in regulating different signaling pathways such as inflammation and apoptosis.However,how linear ubiquitination regulates antiviral immunity remains largely unknown.The purpose of this study is to study the effects of linear ubiquitination on interferon(IFN)-mediated signaling pathway and antiviral function,and to further clarify its specific regulatory mechanism.Furthermore,this project will explore how viral infection regulates linear ubiquitination.Moreover,the significance of linear ubiquitination for IFN signaling pathway will be confirmed by knockout mice.Method:(1)Mass spectrometry was performed to identify the potential interacting proteins of STAT1.Western Blot was used to analyze the effects of linear ubiquitin E3 ligase complex LUBAC(HOIP/HOIL-1L/Sharpin)on linear ubiquitination of STAT1 protein(2)Cells were transfected with LUBAC-overexpressing plasmids or HOIP-knockdown plasmids,and then stimulated with IFN-I.The activation of key proteins in the IFN-I signaling pathway was analyzed by RT-qPCR and Western Blot.The effects of linear ubiquitination on the expression levels of ISGs mRNA were analyzed by RT-qPCR.In addition,LUBAC-mediated regulation of cellular antiviral ability was analyzed by RT-qPCR.(3)By constructing a series of STAT1 lysine mutants,we would determine the key sites of the linear ubiquitination on STAT1.The effects of STAT1 linear ubiquitination mutants on IFN-I signaling pathway were analyzed by Western Blot or RT-qPCR.(4)Through the co-immunoprecipitation(co-IP)experiments,the effects of linear ubiquitination on the interaction between STAT1 and other IFN-I signaling proteins were analyzed,and the detailed mechanism by which STAT1 linear ubiquitination regulated IFN-I signaling pathway were explored(5)Under IFN-I stimulation,the effects of IFN-I on STAT1 linear ubiquitination were analyzed by Western Blot(6)The effects of viral infection on the expression of HOIP and the regulation of STAT1 linear ubiquitination were analyzed by viral infection experiments(7)The effects of the linear ubiquitination modification on IFN signaling and antiviral function were confirmed in knockout mice.Results:(1)STAT1 interacts with HOIP.LUBAC overexpression significantly upregulates the linear ubiquitination of STAT1.However,knockdown of HOIP remarkably inhibits STAT1 linear ubiquitination.(2)Linear ubiquitination restricts STAT1 activation and maintains cellular IFN-I signaling homeostasis(3)LUBAC induces STAT1 linear ubiquitination at Lys511 and Lys652.The mutation of STAT1 linear ubiquitination promotes phosphorylation and activation of STAT1 in IFN-I signaling(4)Linear ubiquitination blocks the binding of STAT1 to IFNAR2,which inhibits the activation of IFN-I signaling pathway.(5)IFN-I stimulation removes STAT1 linear ubiquitination via OTULIN for IFN-I signaling activation.(6)Viral infection upregulates HOIP expression to enhance STAT1 linear ubiquitination,which negatively regulates IFN-I antiviral response.(7)Linear ubiquitination deficiency regulates IFN-I antiviral activity in vivo.Conclusion:The transcription factor STAT1 undergoes linear ubiquitination modifications.Linear ubiquitination inhibits the binding of STAT1 to IFN-I receptor IFNAR2,thus restricting activation of STAT1,which eventually maintains IFN-STAT1 signaling homeostasis.Linear ubiquitin chains of STAT1 are removed by OTULIN in IFN-I signaling,so that STAT1 is able to be activated by IFN-I stimulation.Viral infection can inhibit IFN-I signaling by stimulating HOIP expression via the NF-?B pathway and inducing STAT1 linear ubiquitination.Taken together,our study suggests that linear ubiquitination is an important mechanism controlling the homeostasis and activation of IFN-STAT1 signaling.