摘要
目的研究卵巢癌结构域蛋白酶-1(ovarian tumor domain-containing protease-1,OTUD1)基因对Ⅰ型干扰素信号通路的影响以及对IFN-α抗病毒效应的影响。方法双荧光素酶报告试验检测OTUD1基因对IFN-α诱导的干扰素刺激应答元件(interferon-stimulated response elements,ISRE)启动子转录活性的影响;实时荧光定量PCR(quantitative real-time PCR,qPCR)检测OTUD1对IFN-α诱导的干扰素刺激基因(interferon-stimulated genes,ISGs)表达水平的影响;Western blot检测OTUD1对干扰素信号通路关键分子表达的调控作用,qPCR和ELISA检测其对IFN-α抑制乙型肝炎病毒复制的影响。结果在HEK293T和Huh7.0细胞中,OTUD1下调了干扰素信号通路下游的ISRE启动子转录活性以及ISG15和ISG56等抗病毒基因的表达。在HEK293T细胞中,OTUD1降低了磷酸化蛋白质酪氨酸激酶(phospho-Jak1,p-JAK1)的蛋白质表达水平,但是OTUD1酶活突变体C320S不能调控ISGs和p-JAK1的表达。在Huh7.0细胞中,OTUD1降低了IFN-α抑制乙型肝炎病毒复制的效应。结论OTUD1通过其泛素化酶活性下调p-JAK1的蛋白质表达水平,抑制干扰素JAK-STAT信号通路及ISGs表达。OTUD1拮抗IFN-α抑制病毒复制的效应可能与干扰素诱导的JAK-STAT信号通路有关。
Objective To investigate the effects of ovarian cancer domain containing protease-1(OTUD1)gene on the typeⅠinterferon signaling pathway,and its impact on the antiviral effects of IFN-α.Methods Dual-luciferase reporter assay was performed to detect the effects of OTUD1 gene on the transcriptional activity of interferon-stimulated response element(ISRE)promoter.Quantitative real-time PCR(qPCR)was used to detect the effects of OTUD1 on IFN-α-induced expression of interferon-stimulated genes(ISGs).The effects of OTUD1 gene on the expression of key proteins in the IFN-αsignal transduction pathway were analyzed by Western blot,and its effects on IFN-α-mediated inhibition of hepatitis B virus replication were detected by qPCR and ELISA.Results In HEK293T and Huh7.0 cells,OTUD1 down-regulated the transcriptional activity of ISRE promoter in the interferon signaling pathway and the expression of antiviral genes such as ISG15 and ISG56.In HEK293T cells,OTUD1 reduced the expression of phospho-Jak1(p-JAK1)at protein level,but the catalytic inactive mutant of OTUD1(C320S)could not regulate the expression of ISGs or p-JAK1.In Huh7.0 cells,OTUD1 antagonized the inhibitory effect of IFN-αon hepatitis B virus replication.Conclusions OTUD1 with the ubiquitination activity can inhibit the interferon JAK-STAT signaling pathway and the expression of ISGs by down-regulating the expression of p-JAK1 protein.OTUD1 antagonizes the effect of IFN-αon viral replication and that may be related to the interferon-induced JAK-STAT signaling pathway.
作者
周明慧
江彦君
宋术义
胡源
Zhou Minghui;Jiang Yanjun;Song Shuyi;Hu Yuan(Key Laboratory of Molecular Biology on Infectious Diseases,Ministry of Education,Chongqing Medical University,Chongqing 400016,China)
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2024年第5期382-389,共8页
Chinese Journal of Microbiology and Immunology
基金
国家重点研发计划(2018YFE0107500)。