The Roles Of UGT1A1 And Its Substrate Bilirubin In Antiviral Innate Immune Response

ObjectiveUDP-glucuronosyltransferase 1A1(UGT1A1)is an important member of the phase II metabolic enzyme family in the human body,which is widely involved in the metabolism and detoxification of a variety of endogenous and exogenous substances.Among them,bilirubin is the major endogenous substrate of UGT1A1.At present,studies on UGT1A1 mainly focus on the effect of its enzymatic activity on drug metabolism,while its role in antiviral innate immunity has not been reported.Our study identified a new function of UGT1A1.UGT1A1 positively regulates the type Ⅰ interferon signaling pathway.We further explored the specific mechanisms by which UGT1A1 and bilirubin affect the type Ⅰinterferon signaling pathway,providing new ideas to improve the clinical antiviral efficacy of type Ⅰ interferon.Methods(1)To analyse the role of UGT1A1 in antiviral innate immunity:The changes of UGT1A1 expression level after viral infection were detected by Real-time qPCR and Western Blot;the antiviral function of UGT1A1 was analyzed by Real-time qPCR,Western Blot and inverted fluorescence microscopy observation.(2)To explore the effect of UGT1A1 on interferon signaling pathway:The UGT1A1 overexpression plasmid,knockdown plasmid(shUGT1A1)and enzyme inactive mutant plasmid(UGT1A1-G308E)were transfected into the cells,and the effect of UGT1A1 on the production of interferon and interferon-stimulated genes(ISGs)was detected by Real-time qPCR.The effect of bilirubin,an endogenous substrate of UGT1A1,on interferon and ISGs production was detected by Real-time qPCR.(3)To identify the target proteins of UGT1A1 and explore the specific mechanism of UGT1A1 in regulating interferon signaling pathway:After overexpression of UGT1Al and stimulation of cells with SeV,the effect of UGT1A1 on the phosphorylation of IRF3,a key protein of interferon production pathway,was detected by Western Blot.Real-time qPCR and Western Blot were used to analyze the target proteins of UGT1A1 in promoting interferon production.How UGT1A1 promoted IRF3 phosphorylation was analyzed by coimmunoprecipitation and Western Blot.(4)To explore the effect of bilirubin on interferon signaling pathway:Cells were treated with bilirubin and interferon at the same time,and the effect of bilirubin on ISGs in the interferon signaling pathway was analyzed by Real-time qPCR and Western Blot.The cells were treated with bilirubin and interferon,and then stimulated with virus,and the effect of bilirubin on interferon-mediated antiviral effect was investigated by Real-time qPCR and Western Blot.Cells were treated with bilirubin and interferon simultaneously,and the effects of bilirubin on the key node proteins p-STAT1,p-JAK1 and p-TYK2 in the interferon signaling pathway were analyzed by Western Blot.The interaction between bilirubin and interferon was analyzed by interaction experiments and molecular docking simulations.The effect of bilirubin on the interaction between interferon and receptor was investigated by cell experiments and in vitro interaction experiments.Results(1)Viral infection upregulates the mRNA and protein levels of UGT1A1.(2)UGT1A1 has broad-spectrum antiviral ability,which can significantly inhibit the infection of a variety of viruses,including RNA viruses SeV,VSV,H1N1 and DNA virus HSV-1,etc.This phenomenon has been verified in a variety of cell lines such as HEK293T,HepG2 and 2fTGH.(3)UGT1A1 promotes the production of virus-induced IFNβ and ISGs independent of its enzymatic activity.Bilirubin,the metabolic substrate of UGT1A1,does not affect the production of virus-induced IFNβ,but significantly inhibits the expression of interferoninduced ISGs.(4)By binding with IRF3,UGT1A1 inhibits the interaction between IRF3 and phosphatase PP2A.thus promoting the phosphorylation of IRF3,and then inducing the production of type Ⅰ interferon.(5)Bilirubin can target interferon and inhibit its binding to interferon receptors,thus inhibiting the activation of interferon signaling pathways,including the activation of p-JAK1,p-TYK2,p-STAT1 and ISGs expression induced by type Ⅰ interferon,and ultimately inhibiting interferon-mediated antiviral effects.ConclusionsViral infection upregulates the expression levels of UGT1A1.UGT1A1 targets IRF3 and inhibits its interaction with phosphatase PP2A,thereby promoting interferon production and exerting broad-spectrum antiviral functions.Bilirubin,an endogenous substrate of UGT1A1,targets interferon and inhibits the interaction between type-Ⅰ interferon and its receptors,thereby suppressing the activation of interferon signaling.This study identified UGT1A1 and bilirubin,two novel molecules that regulate the type Ⅰ interferon signaling pathway,providing new ideas to enhance the clinical antiviral efficacy of type Ⅰ interferon.