胞内形式OPN通过稳定TRAF3正向调节抗病毒免疫反应

发布时间：2018-05-12 11:23

  本文选题：iOPN + Ⅰ型干扰素　； 参考：《山东大学》2016年博士论文

【摘要】：固有免疫系统作为机体抵御外界病原体入侵的首道防线,不仅可以激活适应性免疫系统,同时也可以直接对入侵的病原体产生强烈的免疫应答,从而杀伤病原体。在病毒入侵时,固有免疫系统可以激活产生多种细胞因子,其中最主要的是I型干扰素(IFNα/β)。固有免疫系统中的多种的模式识别受体(PRRs)都与Ⅰ型干扰素的产生相关,包括诸如Toll样受体(TLRs), RIG-I样受体(RLRs)和内源性DNA感受器等。产生的Ⅰ型干扰素可以进而与其受体结合,活化JAK (Janus kinase)和STAT (Signaltransducers and activators of transcription)信号通路,进而诱导IFN-stimulated genes (ISGs)的表达,最终清除病毒。骨桥蛋白(osteopontin, OPN)又称为早期T淋巴细胞活化因子1(early T lymphocyte activation 1, Etal),是一种分泌性的多功能糖蛋白。OPN可以调节多种生物体进程,包括细胞分化、粘附、骨重构、恶性肿瘤和免疫反应。长期以来,OPN被认为是一个与炎症过程有关的潜在的促炎症细胞因子,具有促进巨噬细胞分泌IFN-γ和IL-12的作用。近年来,随着胞内形式OPN (iOPN)的发现,OPN在不同免疫细胞以及免疫反应的不同阶段中的不同调控作用逐渐引起人们的重视。而iOPN在固有免疫系统当中尤其是抗病毒免疫当中的作用还未明确。研究目的：探讨OPN,尤其是iOPN是否可以调节病毒诱导的Ⅰ型干扰素产生。如若可以调节Ⅰ型干扰素产生,阐述其潜在机制。研究方法：1.首先检测不同病毒及刺激剂对OPN表达的影响。再利用OPN缺陷型(Spp1-/-)小鼠与野生型(WT)小鼠的腹腔巨噬细胞,经病毒刺激后,检测IFNβ以及下游多种代表性ISGs的mRNA水平变化、蛋白分泌水平变化等。通过构建全长型OPN (full length OPN)及胞内形式OPN(iOPN)表达质粒并转染进HEK293细胞,利用双荧光素酶报告基因,检测两种不同形式OPN对于调节RNA病毒SeV所活化的IFN表达的影响,并检测iOPN质粒对于各种接头分子所活化的IFN启动子区活性的调节作用。2.利用OPN缺陷型(Sppl-/-)小鼠与野生型(WT)小鼠,通过腹腔注射VSV病毒,检测VSV病毒在小鼠脏器中的滴度与含量,反应两类小鼠对于VSV病毒的抵抗能力的区别。3.通过利用OPN缺陷型(Spp1-/-)小鼠与野生型(WT)小鼠腹腔巨噬细胞,以及iOPN过表达质粒转染HEK293细胞,检测经SeV刺激后,重要的IFN转录因子IRF3的活化水平变化。4.经实时荧光定量PCR (q-PCR)以及双荧光素酶报告基因实验,检测iOPN质粒的靶点分子,并经免疫共沉淀(co-IP)以及免疫荧光等实验检测iOPN与靶点分子的结合情况。5.明确靶点分子后,利用不同类型的泛素分子质粒,共转染iOPN及靶点分子进HEK293细胞,检测其是否发生泛素化水平的变化,以及靶点分子的稳定性是否发生变化。6.靶点分子若发生K48位泛素化水平的变化以及其稳定性若发生变化,说明iOPN可能可以调节靶点分子的稳定性。通过对靶点已知的泛素化酶以及去泛素化酶进行筛选,寻找可能的作用机制,并通过细胞水平以及体外翻译系统对结论进行实验性确认。7.通过利用细胞过表达载体以及慢病毒表达载体构建iOPN-WT、iOPN-N、iOPN-C重组过表达载体,通过体外翻译系统实验或对OPN缺陷型小鼠腹腔巨噬细胞进行iOPN回补实验,明确iOPN具体发生功能的区域。研究结果：1.首先,在利用RNA病毒VSV(水泡性口炎病毒)、SeV(仙台病毒)以及DNA病毒HSV(单纯疱疹病毒)等病毒刺激野生型小鼠腹腔巨噬细胞不同时间点后,发现OPN的表达都有逐渐升高的趋势；其次,在OPN缺陷型小鼠以及野生型小鼠巨噬细胞中加入SeV或VSV刺激不同时间后,发现相比较于野生型组,OPN缺陷型小鼠腹腔巨噬细胞产生IFNβ的能力都明显降低；将构建成功的全长型OPN表达载体与iOPN表达载体分别转染进HEK293后经SeV刺激后,发现二种质粒都可以使IFNβ表达增强,但是iOPN表达载体组明显强于全长型OPN表达载体组,并且利用OPN封闭抗体封闭分泌型OPN后,IFNβ的产生无明显变化,说明全长型OPN所表达的分泌性OPN可能并不参与调节IFNβ的增强过程；iOPN也可以明显增强RIG-I、MDA5、TRIF以及STINGcGAS等接头分子所介导的IFNβ启动子区的活化。2.OPN缺陷型小鼠相比较于野生型小鼠,会产生较少的IFNβ,进而造成此组巨噬细胞或成年小鼠在经VSV病毒感染后,VSV病毒的滴度以及VSV mRNA水平以及VSV病毒的蛋白表达程度更高。并且OPN缺陷型小鼠组在大剂量VSV病毒感染后,生存率明显低于野生型小鼠组。3.前述实验结果证明OPN,尤其是iOPN的确可以正调IFNβ的产生,又由于IRF3是重要的调控IFNβ表达的转录因子,我们进而研究IRF3的活化水平是否有变化。我们发现无论是在启动子区结合、磷酸化、二聚化以及入核等方面,iOPN都可以起到正调作用,说明iOPN的确是通过影响IRF3转录因子的活化进而增强了IFNβ的表达。4.为了明确iOPN作用的靶点,我们经报告基因以及realtime-PCR等技术发现iOPN作用的靶点可能存在于MAVS以及TBKl附近。通过免疫共沉淀我们发现iOPN可以特异性与TRAF3结合,并且这一结论也经免疫荧光以及体外蛋白结合实验验证。5.在知道作用靶点为TRAF3后,我们进一步研究可能的机制。泛素化调节作用在机体中具有举足轻重的作用,我们首先研究下iOPN是否会影响TRAF3的泛素化。结果发现,iOPN可以抑制TRAF3发生K48位泛素化修饰,即iOPN可以起到稳定TRAF3表达的作用。无论是在HEK293细胞中过表达iOPN,或是在OPN缺陷型的小鼠腹腔巨噬细胞,iOPN的过表达或缺失都会导致放线菌酮(CHX)介导的TRAF3的稳定性发生明显变化。6.由于我们发现iOPN可以抑制TRAF3发生K48位泛素化修饰,但由于iOPN本身并没有去泛素化酶功能,这就提示我们可能iOPN可以抑制某种可介导TRAF3的K48位泛素化作用的E3连接酶与其结合,或者募集某种可以去除TRAF3的K48位泛素化链的去泛素化酶与TRAF3结合,结果我们发现之前有报道过的TRAF3的去泛素化酶USP25并没有因为iOPN的加入而更加明显的除去TRAF3上的泛素化链,而Triad3A对于TRAF3的K48位泛素化修饰作用则由于iOPN的加入而明显受到抑制。进而我们发现iOPN可以明显的抑制TRAF3与Triad3A的结合,并且TRAF3的Y440、Q442位点如若被突变则TRAF3不与Triad3A结合,同时也不与iOPN结合。综上说明iOPN可能是通过结合了TRAF3进而抑制了Triad3A与TRAF3的结合,iOPN与Triad3A肯能是竞争结合关系。7.通过构建的iOPN-WT以及iOPN-N端或iOPN-C端截短体,我们发现iOPN的C端可以与TRAF3发生结合,并且通过在OPN缺陷型小鼠腹腔巨噬细胞中进行iOPN回补实验,我们进一步确定iOPN的C端即足以发挥结合TRAF3、抑制TRAF3的K48位泛素化以及增强IFNβ产生的作用。总结：1.OPN的表达可被病毒所诱导,并且OPN尤其是iOPN可以正调I型干扰素的产生。2.OPN在体内也是Ⅰ型干扰素产生的强力正调者,可以诱导很强的抗病毒免疫应答。3. iOPN可以正调IRF3转录因子的活化。4. iOPN可以与重要接头分子TRAF3特异性结合。5. IOPN可以调节TRAF3的K48位偶联的泛素化修饰并且使TRAF3更稳定。6. iOPN与Triad3A竞争性结合TRAF3,进而影响Triad3A介导的,TRAF3的K48位偶联的泛素化。7. iOPN通过C端与TRAF3结合,并且其C端即可影响TRAF3的泛素化即稳定性。创新点及意义：1.本研究首次明确完善地证明iOPN可以参与调控抗病毒天然免疫反应,可以正向调节IFNβ的产生以及具有明显的抗病毒功能。这一功能的机制是由于iOPN通过抑制Triad3A与TRAF3的结合,从而抑制了Triad3A对TRAF3的K48位泛素化修饰作用,进而导致TRAF3的稳定性增强,IFNβ的活化进而增强。2.本研究提出iOPN可以抑制某分子的泛素化作用这一机制在国际上尚属少见,极大丰富了我们对于OPN功能的理解。并且我们首次发现iOPN的C端可以发挥几乎全部的抗病毒功能。3.本研究为正向调控抗病毒免疫信号通路提供了新的实验证据,为抗病毒研究以及新药物靶点的研究提供了新的思路。
[Abstract]:As the first line of defense against the invasion of external pathogens, the innate immune system can not only activate the adaptive immune system, but also produce a strong immune response to the invading pathogen, thus killing the pathogen. In the case of virus invasion, the inherent immune system can activate a variety of cytokines, the most important of which are the most important It is type I interferon (IFN alpha / beta). A variety of pattern recognition receptors (PRRs) in the inherent immune system are associated with the production of type I interferon, including such as Toll like receptor (TLRs), RIG-I like receptor (RLRs) and endogenous DNA receptor. Nsducers and activators of transcription) signaling pathway, which then induces the expression of IFN-stimulated genes (ISGs), and eventually clears the virus. The osteopontin (osteopontin, OPN) is also called the early T lymphocyte activation factor 1 (early), a secretory multifunctional glycoprotein that can regulate a variety of raw materials. Process, including cell differentiation, adhesion, bone remodeling, malignant tumor and immune response. OPN has long been thought to be a potential pro-inflammatory cytokine associated with inflammatory processes, which promotes the secretion of IFN- gamma and IL-12 by macrophages. In recent years, with the discovery of intracellular form OPN (iOPN), OPN is in different immune cells and in different immune cells. The different regulatory roles in the different stages of the immune response have gradually aroused people's attention. And the role of iOPN in the inherent immune system, especially in antiviral immunity, is not clear. The purpose of this study is to explore whether OPN, especially iOPN, can regulate the production of virus induced interferon I. The potential mechanisms are discussed. 1. first, the effects of different viruses and stimulants on the expression of OPN were detected. Then the peritoneal macrophages of OPN deficient (Spp1-/-) mice and wild type (WT) mice were used to detect the changes in the mRNA level of IFN beta and a variety of representative ISGs, and the changes of protein secretion level. The full length OPN (full length OPN) and intracellular form OPN (iOPN) expressed plasmid and transfected into HEK293 cells, using the double luciferase reporter gene to detect the effect of two different forms of OPN on the regulation of IFN expression of the activation of RNA virus SeV, and to detect the regulation of the iOPN plasmids on the activation of the promoter region activated by various connector molecules. 2. using OPN deficient (Sppl-/-) mice and wild type (WT) mice, the titer and content of VSV virus in the mouse organs were detected by intraperitoneal injection of VSV virus. The difference of the resistance ability of the two types of mice to the resistance of VSV virus.3. was expressed by using OPN deficient (Spp1-/-) mice and wild type (WT) mice peritoneal macrophages, and iOPN overexpression. HEK293 cells were transfected by plasmid, and the activation level of the important IFN transcription factor IRF3 was detected by SeV stimulation..4. was detected by real-time fluorescent quantitative PCR (q-PCR) and double luciferase reporter gene test, and the target molecules of iOPN plasmids were detected, and the binding of iOPN and target molecules was detected by immunoprecipitation (co-IP) and immunofluorescence. 5. after identifying the target molecules, using different types of ubiquitin plasmids, CO transfection of iOPN and target molecules into HEK293 cells to detect the changes in the level of ubiquitination, and whether the stability of the target molecules changes if the.6. target molecule changes the level of K48 ubiquitination and its stability changes, indicating iOP N may be able to regulate the stability of the target molecules. Through the screening of ubiquitin and ubiquitination enzymes that are known to the target, the possible mechanisms are found, and the results are experimentally confirmed by the cell level and in vitro translation system, and.7. is constructed by using cell overexpressed carrier and lentivirus expression vector to construct iOPN-WT, iOPN- N, iOPN-C recombinant overexpression vector, through the in vitro translation system experiment or the OPN deficient mouse peritoneal macrophages to carry out the iOPN recharge experiment to clarify the specific region of iOPN function. 1. first, in the use of RNA virus VSV (vesicular stomatitis virus), SeV (Sendai virus) and DNA virus HSV (herpes simplex virus) and other virus spines. After different time points of peritoneal macrophages in wild type mice, it was found that the expression of OPN had a tendency to increase gradually. Secondly, after adding SeV or VSV to the macrophages of OPN deficient mice and wild type mice for different time, it was found that the ability to produce IFN beta in the peritoneal macrophages of OPN deficient mice was better than that in the wild type. The whole long OPN expression vector and iOPN expression vector were transfected into HEK293 respectively. After SeV stimulation, it was found that all two plasmids could enhance the expression of IFN beta, but the iOPN expression vector group was obviously stronger than the full length OPN expression vector group, and the production of IFN beta was not obvious after the OPN closed antibody closed the secretory OPN. It shows that the secretory OPN expressed by the full-length OPN may not be involved in the regulation of the enhancement of IFN beta; iOPN can also significantly enhance the activation of the.2.OPN deficient mice in the IFN beta promoter region mediated by RIG-I, MDA5, TRIF, and STINGcGAS. After VSV virus infection, the titer of the VSV virus and the level of VSV mRNA and the protein expression of the VSV virus are higher in the phagocytic or adult mice. The survival rate of the OPN deficient mice group in the large dose VSV virus infection is significantly lower than that of the wild type mouse group.3. before the experimental results of.3., especially that iOPN can actually be adjusted IFN beta. Because IRF3 is an important transcription factor that regulates the expression of IFN beta, we further investigate whether there is a change in the activation level of IRF3. We found that iOPN can play a positive role in the activation of the promoter region binding, phosphorylation, dimerization, and nucleation, indicating that iOPN does increase by the activation of the IRF3 transcription factor. The expression of IFN beta (.4.) is stronger to identify the target of iOPN action. The target of our reporter gene and realtime-PCR technology may be found near MAVS and TBKl. By immunoprecipitation, we found that iOPN can be specifically associated with TRAF3, and this conclusion is also tested by immunofluorescence and in vitro protein binding experiments. We further study the possible mechanism of.5. after we know that the target target is TRAF3. The regulation of ubiquitination plays an important role in the body. We first study whether iOPN will affect the ubiquitination of TRAF3. It is found that iOPN can inhibit the K48 ubiquitination of TRAF3, that is, iOPN can stabilize TRAF3 expression. Effect. Either over expression of iOPN in HEK293 cells, or in OPN deficient mice peritoneal macrophages, the overexpression or deletion of iOPN causes significant changes in the stability of TRAF3 mediated by actinomycone (CHX),.6. because we found iOPN can inhibit the K48 bit ubiquitination of TRAF3, but because iOPN itself is not ubiquitous The function of the protease suggests that iOPN may inhibit the binding of a E3 ligase that mediates the ubiquitination of the K48 site of TRAF3, or collects a K48 bit ubiquitinating chain that removes TRAF3 by binding to TRAF3. As a result, we found that the previously reported TRAF3's de ubiquitinase USP25 was not due to I. The addition of OPN is more obvious to remove the ubiquitination chain on TRAF3, and the ubiquitination of K48 in TRAF3 is obviously inhibited by the addition of iOPN. Furthermore, we find that iOPN can obviously inhibit the combination of TRAF3 and Triad3A, and TRAF3 Y440, if the Q442 position is mutated, iOPN can not be combined with the Triad3A. We do not combine with iOPN. It shows that iOPN may be combined with TRAF3 to inhibit the combination of Triad3A and TRAF3, iOPN and Triad3A can be a competitive combination of.7. through the construction of iOPN-WT and iOPN-N end or iOPN-C truncate. IOPN recuperate in the cavity of macrophages, we further confirm that the C end of iOPN is sufficient to play the role of binding TRAF3, inhibiting K48 ubiquitination of TRAF3 and enhancing IFN beta production. It is concluded that the expression of 1.OPN can be induced by the virus, and OPN especially iOPN can regulate I type of interferon in the body as well as type I interferon The strong positive regulator can induce a strong anti-virus immune response,.3. iOPN can regulate the activation of the IRF3 transcription factor,.4. iOPN can be combined with the TRAF3 specificity of the important joint molecule,.5. IOPN can modulate the ubiquitination of TRAF3 K48 bit coupling and make TRAF3 more stable.6. Triad3A mediated, TRAF3 K48 coupled ubiquitination.7. iOPN is combined with TRAF3 through C terminal, and its C end can affect the ubiquitination of TRAF3 as stability. Innovation and significance: 1. this study first clearly and perfectly demonstrated that iOPN can participate in the regulation of antiviral natural immune responses, with positive regulation of the production of IFN beta and obvious The mechanism of this function is that the mechanism of this function is that iOPN inhibits the binding of Triad3A to TRAF3 by inhibiting the ubiquitination of K48 in TRAF3 by Triad3A, and thus leads to the stability of TRAF3, and the activation of IFN beta enhances the.2. based study that the mechanism that iOPN can inhibit the ubiquitination of a molecule is still internationally It is rare, which greatly enriches our understanding of OPN function, and we first found that the C end of iOPN can play almost all the antiviral function.3.. This study provides new experimental evidence for the positive regulation of antiviral immune signaling pathway, which provides new ideas for the research of antiviral and new drug targets.

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R392
，

本文编号：1878427