E3泛素连接酶RNF2在Ⅰ型干扰素抗病毒天然免疫反应中的调控功能及其机制研究

发布时间：2017-12-28 08:41

本文关键词：E3泛素连接酶RNF2在Ⅰ型干扰素抗病毒天然免疫反应中的调控功能及其机制研究　出处：《北京协和医学院》2017年博士论文　论文类型：学位论文

【摘要】：天然免疫系统介导的抗病毒反应是机体抵抗病毒侵袭的第一道防线。抗病毒天然免疫反应的不足或过强,都会导致相关疾病的发生,因此对抗病毒天然免疫反应的调控机制研究具有重要的意义。翻译后修饰(Post-translational modification,PTM)在抗病毒天然免疫反应中具有重要的调控作用,其中由E3泛素连接酶介导的泛素化修饰是必不可少的一种,但是含有RING结构域的E3家族(RING E3s)在抗病毒天然免疫反应中的功能还没有被完全解析。Ⅰ型干扰素(Type Ⅰ interferon,IFN-Ⅰ)信号通路的激活是抗病毒天然免疫反应的中心环节,由转录因子 STAT1(Signal transducer and activator of transcription 1)介导产生的一系列干扰素刺激基因(Interferon-stimulated genes,ISGs)则最终决定了抗病毒反应的效应。然而,过度激活的IFN-Ⅰ/STAT1信号不仅不能够维持抗病毒效应,还会引起免疫系统的抑制,导致慢性的病毒感染。因此,对IFN-Ⅰ/STAT1信号通路的负向调控机制研究对于IFN-Ⅰ的临床应用具有重要的价值。为寻找抗病毒天然免疫反应新的调控分子,解析RING E3家族在抗病毒天然免疫反应中的作用及机制,我们对小鼠的115个RING E3基因进行了以siRNAs(Small interfering RNAs)干扰为基础的高内涵筛选。通过与对照干扰的结果相比较,我们共鉴定到9个正向的调控分子及30个负向调控分子。为进一步寻找IFN-Ⅰ/STAT1信号的新的负向调控分子,我们对筛选到的30个负向调控分子进行了第二轮筛选。通过外源加入IFN-β后,我们发现有9个分子仍然表现出负向调控的作用,说明这9个分子主要通过对IFN-Ⅰ/STAT1信号的负向调控来实现对抗病毒天然免疫反应的负向调控。在这9个分子中,我们选取了具有显著效应的ring finger protein 2(RNF2)进行了详细的功能和机制研究。通过体外干扰,细胞和小鼠水平的基因敲除,细胞水平的过表达等一系列试验,我们发现RNF2无论是体外还是体内,均能够负向调控VSV或IFN-Ⅰ刺激的ISGs的表达,从而抑制细胞和小鼠对病毒的抵抗能力。通过相互作用质谱及泛素化修饰定量质谱,我们鉴定了 15个RNF2在抗病毒反应中的潜在底物,其中包括转录因子STAT1,同时我们还鉴定到了 STAT1上可能受RNF2调控的12个潜在赖氨酸位点。通过免疫沉淀和体外GST pull down的方法,我们证明了RNF2与STAT1的直接相互作用,并且通过转染一系列的RNF2或STAT1截短体,泛素分子突变体后进行免疫沉淀实验,我们发现RNF2主要通过其RING结构域增强IFN-Ⅰ刺激下STAT1的K33位连接的多聚泛素化修饰,并抑制了 STAT1介导的启动子序列ISRE的转录活性。在机制的研究中,我们利用染色质免疫沉淀,体外的DNA pull down等实验发现RNF2并不影响IFN-Ⅰ/STAT1信号通路的激活,而是通过修饰STAT1的DNA结合区域的K379位点促进STAT1与DNA的解离,间接促进其发生去磷酸化反应,从而及时抑制IFN-Ⅰ/STAT1信号。我们的研究不仅系统性地解析了 RING E3分子在抗病毒天然免疫领域内的调控功能,还发现了一系列抗病毒天然免疫反应的新的调控分子,更为IFN-Ⅰ/STAT1信号通路的负向调控提供了新的机制。因此,我们的研究结果具有临床指导和转化价值,不仅为治疗病毒感染提供了新的潜在靶点和思路,为新的抗病毒药物的开发奠定了理论基础,也为增强IFN-Ⅰ的治疗效果,防止IFN-Ⅰ过度活化或长期应用造成的免疫耐受、免疫抑制甚至免疫损伤提供了新的理论依据和诊断治疗靶标。
[Abstract]:The antiviral reaction mediated by the natural immune system is the first line of defense against the invasion of the virus. Inadequate or excessive antiviral innate immune responses can lead to the occurrence of related diseases. Therefore, the research on the regulation mechanism of the natural immune response against viruses is of great significance. Post translational modifications (Post-translational modification, PTM) play an important role in antiviral innate immune responses, including ubiquitination mediated by the E3 ubiquitin ligase is an essential, but the RING domain containing E3 (RING E3s) family in the anti viral innate immune response function has not been fully resolved. Type I interferon (Type 1 interferon IFN- 1) signal pathway is a central link of antiviral innate immune response, the transcription factor STAT1 (Signal transducer and activator of transcription 1) a series of IFN mediated stimulation by gene (Interferon-stimulated genes ISGs) effect is the final decision of the antiviral response. However, the excessively activated IFN- I /STAT1 signal not only does not maintain the antiviral effect, but also causes the suppression of the immune system, resulting in chronic viral infection. Therefore, the study of the negative regulation mechanism of IFN- I /STAT1 signaling pathway is of great value for the clinical application of IFN- I. In order to find new regulatory molecules for the antiviral innate immune response, we analyzed the function and mechanism of RING E3 family in the antiviral innate immune response. We carried out a high content screening based on siRNAs (Small interfering RNAs) interference to 115 RING E3 genes in mice. By comparing with the results of control interference, we have identified 9 positive regulators and 30 negative regulators. In order to further search for new negative regulator molecules of IFN- I /STAT1 signal, we screened 30 negative regulator molecules by second rounds. After exogenous IFN- beta addition, we found that 9 molecules still showed negative regulation role, indicating that these 9 molecules were mainly through negative regulation of IFN- I /STAT1 signal to achieve negative regulation of the innate immune response against virus. In these 9 molecules, we have selected the ring finger protein 2 (RNF2) with significant effects to carry out a detailed study of the functions and mechanisms. Through in vitro interference, cells and mouse levels of gene knockout, cell level expression and a series of experiments, we found that RNF2 both in vitro and in vivo, can negatively regulate the expression of VSV or IFN- 1 stimulation ISGs, thereby inhibiting cell and mice resistance to virus. Through interaction mass spectrometry and ubiquitination modified mass spectrometry, we identified 15 potential substrates for RNF2 in the antiviral response, including transcription factor STAT1. Meanwhile, we identified 12 potential lysine sites that might be regulated by RNF2 on STAT1. By the method of immunoprecipitation and in vitro GST pull down, we demonstrate that the direct interaction between RNF2 and STAT1, and through a series of RNF2 or STAT1 transfected with truncated mutant ubiquitin molecules, after immunoprecipitation experiments, we found that RNF2 mainly through its RING domain by STAT1 K33 under the stimulation of IFN- I connect poly ubiquitination, and inhibits the transcriptional activity of the promoter sequence of STAT1 mediated ISRE. In the mechanism study, we used chromatin immunoprecipitation, DNA pull down in vitro found that RNF2 did not affect the activation of the IFN- of the /STAT1 signaling pathway, but dissociation by modifying the STAT1 DNA binding sites of STAT1 and K379 to promote DNA, indirectly promote the phosphorylation reaction to occur, thereby inhibiting IFN- I /STAT1 the signal in time. Our research not only systematically analyzed the regulatory function of RING E3 molecule in the field of antiviral innate immunity, but also discovered a series of new regulatory molecules of natural immune response to antiviral, which provided a new mechanism for the negative regulation of IFN- I /STAT1 signaling pathway. Therefore, our results have clinical guidance and value transformation, not only provides a new potential target and ideas for the treatment of viral infections, which laid a theoretical foundation for the development of new antiviral drugs, but also to enhance the therapeutic effect of IFN- I, IFN- I prevent the excessive activation caused by long-term application of immune tolerance, immune suppression even the immune injury provides a theoretical basis and a new target for diagnosis and treatment.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R392

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