NLRX1抑制LUBAC介导的NF-κB激活的机制研究

发布时间：2018-06-25 21:09

本文选题：NLRX1 + LUBAC　；参考：《华中农业大学》2017年硕士论文

【摘要】：蛋白翻译后的泛素化修饰在调节各种细胞生物功能的过程中发挥着关键的作用。近几年新发现的线性泛素链组装复合体(linear ubiquitin chain assembly complex,LUBAC)由HOIP、HOIL-1L和SHARPIN组成,能够产生一种新型的Met1连接的线性多聚泛素链,可以特异性调控经典的NF-κB信号通路,是天然免疫反应的重要组成部分。NLRs是一类重要的胞浆型模式识别受体(PRR),负责识别细胞内信号,是固有免疫针对病原微生物的一类重要感受器。NLRX1是NLRs家族中唯一定位于线粒体的成员,结构较为保守,能够通过多种途径参与NF-κB信号通路的调控,而NLRX1对LUBAC介导的NF-κB信号通路的调控作用尚未有人报道。因此,本论文以NLRX1为研究对象,初步探讨了其对LUBAC介导的NF-κB信号通路的影响,并进一步解释其作用机制。主要研究内容如下:1.NLRX1抑制LUBAC诱导的NF-κB启动子活化在HEK-293T细胞中,分别转染LUBAC、NF-κB-Luc、pRL-TK和不同剂量的NLRX1真核表达质粒,双荧光素酶报告系统检测发现NLRX1能够显著抑制LUBAC激活的NF-κB启动子活性,并且呈剂量依赖性。说明NLRX1能够负调控LUBAC介导的NF-κB信号通路。2.从互作角度探究NLRX1和LUBAC的关系LUBAC由HOIL-1L、HOIP和SHARPIN组成,为了探究NLRX1负调控LUBAC介导的NF-κB信号通路的作用机制,我们首先从互作角度探究NLRX1和LUBAC的关系。通过免疫共沉淀实验(CO-IP)发现,超表达条件下NLRX1与HOIL-1L、HOIP和SHARPIN均存在相互作用,但生理条件下,NLRX1只与HOIP存在互作,且NLRX1是通过其NOD结构域与HOIP的UBA结构域互作;同时还发现NLRX1能够抑制LUBAC复合物的形成,减少LUBAC对NEMO的招募。3.NLRX1发挥负调控作用的关键结构域为LRR结构域为了探究NLRX1发挥负调控作用的关键结构域,构建了NLRX1的4个截短突变体,NLRX1-N-ter(1-468bp)、NLRX1-ΔN-ter(469-2928bp)、NLRX1-LRR(976-2928bp)和NLRX1-ΔLRR(1-975bp)。用双荧光素酶报告系统分析,结果发现NLRX1的LRR结构域在负调控LUBAC依赖的NF-κB通路中发挥着重要作用。4.NLRX1对LUBAC定位及表达的影响利用间接免疫荧光实验(IFA)检测NLRX1对LUBAC定位的影响,发现NLRX1和LUBAC存在共定位且定位于胞浆,NLRX1对LUBAC的定位几乎没有影响。荧光定量PCR实验表明NLRX1也不影响LUBAC的mRNA水平;Western Blot检测发现NLRX1特异性抑制HOIP的表达,且呈剂量依赖性,但用MG132处理后,与对照组相比,HOIP的表达得到恢复,表明NLRX1介导的HOIP的降解是通过蛋白酶体途径实现的。5.NLRX1抑制NEMO的线性泛素化修饰在LUBAC介导的NF-κB信号通路中,LUBAC能够招募NEMO并对其进行线性泛素化修饰,引起IKK复合物的活化及IκBα的降解,促使NF-κB的释放和入核。为了验证NLRX1降解HOIP的生物学意义,通过免疫共沉淀实验检测NLRX1对内源性NEMO的线性泛素化的影响,结果发现NLRX1能够抑制内源性NEMO的线性泛素化修饰,进而负调控LUBAC介导的NF-κB信号通路。
[Abstract]:Ubiquitin modification after protein translation plays a key role in regulating the biological functions of various cells. The newly discovered linear ubiquitin chain assembly complex (linear ubiquitin chain assembly complexLUBAC) is composed of HOIPHIOIL-1L and SHARPIN. It can produce a new type of Met1-linked linear polyubiquitin chain, which can specifically regulate the classical NF- 魏 B signaling pathway. NLRs are important cytosolic pattern recognition receptors (PRR) that recognize intracellular signals. NLRX1 is the only member of the NLRs family that is located in mitochondria, and its structure is conserved, and it can participate in the regulation of NF- 魏 B signaling pathway through many ways. The regulation of NLRX1 on LUBAC mediated NF- 魏 B signaling pathway has not been reported. Therefore, NLRX1 is taken as the research object in this paper. The effects of NLRX1 on the NF- 魏 B signaling pathway mediated by LUBAC are preliminarily discussed, and the mechanism of NLRX1 is further explained. The main results were as follows: 1. NLRX1 inhibited the activation of NF- 魏 B promoter induced by LUBAC in HEK-293T cells and transfected LUBAC- 魏 B-LucpRL-TK and different doses of NLRX1 eukaryotic expression plasmids respectively. The double luciferase reporting system showed that NLRX1 could significantly inhibit the activity of NF- 魏 B promoter activated by LUBAC. And in a dose-dependent manner. NLRX1 can negatively regulate the NF- 魏 B signaling pathway mediated by LUBAC. To explore the relationship between NLRX1 and LUBAC from the perspective of interaction, LUBAC is composed of HOIL-1LHIP and SHARPIN. In order to explore the mechanism of NLRX1 negatively regulating the NF- 魏 B signaling pathway mediated by LUBAC, we first explored the relationship between NLRX1 and LUBAC from the perspective of interaction. The results of co-immunoprecipitation assay (CO-IP) showed that NLRX1 interacted with HOIL-1LLHIP and SHARPIN under over-expression condition, but NLRX1 only interacted with HoIP under physiological conditions, and NLRX1 interacted with HoIP's UBA domain through its nod domain. It was also found that NLRX1 could inhibit the formation of LUBAC complex and reduce the recruitment of Nemo by LUBAC. 3. The key domain of NLRX1 to play a negative regulatory role was LRR domain. Four truncated mutants of NLRX1, NLRX1-N-ter (1-468bp), NLRX1- 螖 N-ter (469-2928bp), NLRX1-LRR (976-2928bp) and NLRX1- 螖 LRR (1-975bp) were constructed. The LRR domain of NLRX1 played an important role in the negative regulation of LUBAC dependent NF- 魏 B pathway. 4. The effect of NLRX1 on LUBAC localization and expression was detected by indirect immunofluorescence assay (IFA). It was found that NLRX1 and LUBAC were colocated and the localization of NLRX1 in cytoplasm had little effect on the localization of LUBAC. Fluorescence quantitative PCR assay showed that NLRX1 did not affect the mRNA level of LUBAC. Western blot analysis showed that NLRX1 specifically inhibited the expression of HoIP in a dose-dependent manner, but the expression of HOIP recovered after treatment with MG132. It is suggested that NLRX1-mediated HOIP degradation is mediated by the proteasome pathway. 5. NLRX1 inhibits the linear ubiquitin modification of Nemo in LUBAC mediated NF- 魏 B signaling pathway, and LUBAC can recruit and modify Nemo by linear ubiquitin modification. Ik complex was activated and I 魏 B 伪 degraded, NF- 魏 B was released and nucleated. In order to verify the biological significance of NLRX1 degradation of hop, the effect of NLRX1 on the linear ubiquitin of endogenous Nemo was detected by immunoprecipitation. The results showed that NLRX1 could inhibit the linear ubiquitin modification of endogenous Nemo. Furthermore, the LUBAC mediated NF- 魏 B signaling pathway was negatively regulated.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.4

【相似文献】