E3连接酶Nrdp1调控巨噬细胞功能及其分子机制研究

发布时间：2019-06-26 20:26

【摘要】：泛素化是由单个或多个泛素在泛素激活酶E1、泛素结合酶E2、及泛素连接酶E3的作用下共价修饰底物蛋白质的过程。研究发现,泛素化不仅可导致底物蛋白质的降解,还可影响底物蛋白质的活性和细胞内定位,是一种调节细胞内蛋白功能和表达水平的重要机制。泛素化修饰可调节细胞的信号转导、分裂和分化等许多过程,同样也在天然免疫以及获得性免疫过程中起重要作用。巨噬细胞是一种重要的免疫细胞,活化的巨噬细胞能分泌大量的酶、细胞因子、NO等活性产物,对于免疫反应的调节和炎症的发展尤为重要。巨噬细胞的活化途径主要有2条,即经典激活途径(classical activation, M1)以及替代激活途径(alternative activation, M2).但泛素化修饰是否参与调控巨噬细胞活化,并没有相关研究。本研究对新型泛素E3连接酶Nrdp1进行了蛋白组学方面的研究。通过DIGE (difference in gel electrophoresis)双向电泳-质谱分析,发现在Nrdp1转基因(Nrdp1-TG)小鼠的腹腔巨噬细胞中,表达较高水平的精氨酸酶1(arginase1, Argl)。Arg1是M2型巨噬细胞极化的标志分子,进一步的研究发现,在IL-4活化的Nrdp1-TG小鼠的腹腔巨噬细胞中,M2型巨噬细胞极化相关的其他分子如Fizzl、Ym1及MR也显著上调。Nrdp1干扰RNA的实验也得到了一致的结果。此外,Nrdp1可有效抑制LPS活化的腹腔巨噬细胞iNOS和炎性细胞因子(IL-6、TNF-α、IL-1β)的产生,但促进抗炎细胞因子IL-10的分泌。为了进一步研究Nrdp1调控Argl表达的分子机制,免疫共沉淀实验结果发现,Nrdp1能够结合Arg基因上游转录因子C/EBPβ,但不能结合STAT6,并且促进IL-4诱导的C/EBPβ泛素化。荧光素酶报告基因实验发现,Nrdp1可促进C/EBPβ诱导的Arg1基因的转录。对Nrdp1-TG小鼠巨噬细胞抗C/EBPβ的免疫沉淀产物进行泛素化抗体免疫印迹检测发现,E3连接酶Nrdp1可有效促进C/EBPβ的泛素化。因此本研究提示,Nrdp1可通过结合并促进C/EBPp的泛素化活化,上调巨噬细胞中Arg1的表达,从而促进巨噬细胞的M2型活化。
[Abstract]:Ubiquitin is a process by which one or more ubiquitin covalently modifies substrate proteins under the action of ubiquitin activating enzyme E1, ubiquitin binding enzyme E2 and ubiquitin ligase E3. It is found that ubiquitin can not only lead to the degradation of substrate protein, but also affect the activity and intracellular localization of substrate protein, which is an important mechanism to regulate the function and expression level of intracellular protein. Ubiquitin modification can regulate many processes such as signal transduction, division and differentiation of cells, and also plays an important role in innate immunity and acquired immunity. Macrophages are an important immune cell. Activated macrophages can secrete a large number of enzymes, cytokines, NO and other active products, which is particularly important for the regulation of immune response and the development of inflammation. There are two main activation pathways of macrophages, namely, classical activation pathway (classical activation, M1 and alternative activation pathway (alternative activation, M2). However, there is no related study on whether ubiquitin modification is involved in the regulation of macrophage activation. In this study, the proteome of a novel ubiquitin E3 ligase Nrdp1 was studied. By DIGE (difference in gel electrophoresis) two-dimensional electrophoresis-mass spectrometry analysis, it was found that the high level of arginine enzyme 1 (arginase1, Argl). Arg1 was a marker of polarization of M2 macrophages in the abdominal macrophages of Nrdp1 transgenic (Nrdp1-TG) mice. Further studies showed that other molecules related to polarization of M2 macrophages, such as Fizzl, were found in the abdominal macrophages of IL-4 activated Nrdp1-TG mice. Ym1 and MR were also significantly up-regulated. The results of Nrdp1 interfering with RNA were consistent. In addition, Nrdp1 can effectively inhibit the production of iNOS and inflammatory cytokines (IL-6,TNF- 伪, IL-1 尾) in abdominal macrophages activated by LPS, but promote the secretion of anti-inflammatory cytokine IL-10. In order to further study the molecular mechanism of Nrdp1 regulating Argl expression, the results of immunoprecipitation assay showed that Nrdp1 could bind to the upstream transcription factor C/EBP 尾 of Arg gene, but could not bind to STAT6, and promote the ubiquitin of C/EBP 尾 induced by IL-4. Luciferase reporter gene assay showed that Nrdp1 could promote the transcription of Arg1 gene induced by C/EBP 尾. The immunoprecipitation products of macrophage anti-C/EBP 尾 in Nrdp1-TG mice were detected by ubiquitin antibody immunoblotting. It was found that E3 ligase Nrdp1 could effectively promote the ubiquitin of C/EBP 尾. Therefore, this study suggests that Nrdp1 can up-regulate the expression of Arg1 in macrophages by binding and promoting ubiquitin activation of C/EBPp, thus promoting M2 activation of macrophages.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R392

【相似文献】