【摘要】：泛素化是由單個(gè)或多個(gè)泛素在泛素激活酶E1、泛素結(jié)合酶E2、及泛素連接酶E3的作用下共價(jià)修飾底物蛋白質(zhì)的過程。研究發(fā)現(xiàn),泛素化不僅可導(dǎo)致底物蛋白質(zhì)的降解,還可影響底物蛋白質(zhì)的活性和細(xì)胞內(nèi)定位,是一種調(diào)節(jié)細(xì)胞內(nèi)蛋白功能和表達(dá)水平的重要機(jī)制。泛素化修飾可調(diào)節(jié)細(xì)胞的信號(hào)轉(zhuǎn)導(dǎo)、分裂和分化等許多過程,同樣也在天然免疫以及獲得性免疫過程中起重要作用。巨噬細(xì)胞是一種重要的免疫細(xì)胞,活化的巨噬細(xì)胞能分泌大量的酶、細(xì)胞因子、NO等活性產(chǎn)物,對(duì)于免疫反應(yīng)的調(diào)節(jié)和炎癥的發(fā)展尤為重要。巨噬細(xì)胞的活化途徑主要有2條,即經(jīng)典激活途徑(classical activation, M1)以及替代激活途徑(alternative activation, M2).但泛素化修飾是否參與調(diào)控巨噬細(xì)胞活化,并沒有相關(guān)研究。 本研究對(duì)新型泛素E3連接酶Nrdp1進(jìn)行了蛋白組學(xué)方面的研究。通過DIGE (difference in gel electrophoresis)雙向電泳-質(zhì)譜分析,發(fā)現(xiàn)在Nrdp1轉(zhuǎn)基因(Nrdp1-TG)小鼠的腹腔巨噬細(xì)胞中,表達(dá)較高水平的精氨酸酶1(arginase1, Argl)。Arg1是M2型巨噬細(xì)胞極化的標(biāo)志分子,進(jìn)一步的研究發(fā)現(xiàn),在IL-4活化的Nrdp1-TG小鼠的腹腔巨噬細(xì)胞中,M2型巨噬細(xì)胞極化相關(guān)的其他分子如Fizzl、Ym1及MR也顯著上調(diào)。Nrdp1干擾RNA的實(shí)驗(yàn)也得到了一致的結(jié)果。此外,Nrdp1可有效抑制LPS活化的腹腔巨噬細(xì)胞iNOS和炎性細(xì)胞因子(IL-6、TNF-α、IL-1β)的產(chǎn)生,但促進(jìn)抗炎細(xì)胞因子IL-10的分泌。為了進(jìn)一步研究Nrdp1調(diào)控Argl表達(dá)的分子機(jī)制,免疫共沉淀實(shí)驗(yàn)結(jié)果發(fā)現(xiàn),Nrdp1能夠結(jié)合Arg基因上游轉(zhuǎn)錄因子C/EBPβ,但不能結(jié)合STAT6,并且促進(jìn)IL-4誘導(dǎo)的C/EBPβ泛素化。熒光素酶報(bào)告基因?qū)嶒?yàn)發(fā)現(xiàn),Nrdp1可促進(jìn)C/EBPβ誘導(dǎo)的Arg1基因的轉(zhuǎn)錄。對(duì)Nrdp1-TG小鼠巨噬細(xì)胞抗C/EBPβ的免疫沉淀產(chǎn)物進(jìn)行泛素化抗體免疫印跡檢測發(fā)現(xiàn),E3連接酶Nrdp1可有效促進(jìn)C/EBPβ的泛素化。因此本研究提示,Nrdp1可通過結(jié)合并促進(jìn)C/EBPp的泛素化活化,上調(diào)巨噬細(xì)胞中Arg1的表達(dá),從而促進(jìn)巨噬細(xì)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- 偽, 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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R392

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