粉尘诱导基因蛋白与细胞内功能蛋白的相互作用及其分子机制的研究

发布时间：2018-05-28 06:00

本文选题：粉尘诱导基因 + 蛋白质组学　；参考：《武汉大学》2016年博士论文

【摘要】：粉尘诱导基因mdig(又称mina53, MINA或N052)是在煤矿工人巨噬细胞中发现的环境诱导基因。mdig的表达升高与多种肿瘤的发病与预后相联系。然而mdig如何作用于细胞代谢和肿瘤发展仍有待阐明。有研究指出mdig蛋白通过其JmjC结构域,参与组蛋白H3在甘氨酸H3K9me3的去甲基化作用。然而不同于其他经典的去甲基化酶,mdig缺乏与染色质接合的蛋白结构域。于是ndig对染色质或组蛋白的作用更可能是通过与其他染色质接合蛋白的相互作用而完成的。第一部分蛋白质组学分析粉尘诱导基因蛋白与细胞内功能蛋白的相互作用目的：本研究旨在通过蛋白质组学分析,发掘细胞中与粉尘诱导基因ndig相互作用的功能蛋白,以探查mdig在细胞代谢和肿瘤发生过程中所起的作用。方法：在人肺癌细胞A549和人支气管上皮细胞BEAS-2B中,运用免疫共沉淀技术分别沉淀mdig和对照组IgG的抗体蛋白复合物。该蛋白复合物在SDS-PAGE胶中电泳分离,用考马斯亮蓝染色观察mdig蛋白的条带模式。在四组独立实验中,应用两种不同的多段高效液相色谱-电喷雾-串联质谱(Orbitrap Fusion和OrbitrapXL)分析与mdig相互结合的蛋白。筛选出的蛋白,用免疫共沉淀结合免疫印迹的方法加以验证。最后用IPA软件统计分析A549细胞和BEAS-2B细胞中与mdig相互作用蛋白的分子通路。结果：在考马斯亮蓝染色的蛋白质分离胶中,三至五条特异性条带稳定的出现在mdig抗体沉淀的蛋白样本中,而在对照组IgG中未被发现。蛋白质谱分析结果显示,在mdig抗体沉淀的蛋白复合物中可检测到一些DNA修复相关蛋白和染色质连接蛋白,包括XRCC5, XRCC6, RBBP4, CBX8, PRMT5,和TDRD等。免疫印迹实验进一步确认了mdig与这些蛋白的结合。在BEAS-2B细胞中,mdig蛋白的相互作用与A549细胞相似,但仍有一些蛋白,如CBX3和CBX5等,仅在BEAS-2B细胞中被观察到与mdig有相互作用。结论：mdig通过与伴侣蛋白的相互作用完成其在表观遗传学、DNA修复、DNA复制和细胞生长调节的功能。第二部分粉尘诱导基因蛋白通过DNA非同源末端接合通路参与DNA损伤修复目的：DNA双链断裂(DSBs)的修复,是在哺乳动物细胞中维持基因组完整性和预防肿瘤形成的关键。蛋白质组学研究发现粉尘诱导基因Mdig与DSBs的非同源末端连接修复(NHEJ)的关键蛋白XRCC6、XRCC5和DNA-PK等有相互结合作用。本研究旨在探索mdig在NHEJ修复通路中所起的作用,并验证mdig通过与功能蛋白XRCC6的相互作用,完成其对DNA损伤修复的影响。方法：建立稳定的mdig过表达细胞系、mdig空载体传染细胞系、mdig shRNA转染的基因沉默细胞系和对照shRNA转染细胞系。分别用30 gM腐草霉素(phleomycin)处理各细胞0,0.25,0.5,1,2,4小时后,应用免疫印迹实验测定各细胞系中NHEJ通路蛋白的变化情况,并应用免疫荧光法观察DNA断裂标记物γH2AX和pDNA-PKcs的表达。彗星实验检测各细胞系对phleomycin处理的DNA损伤敏感程度。结果：Mdig可与DNA损伤的非同源末端接合修复(NHEJ)蛋白XRCC6相互作用,并影响NHEJ通路的关键酶pDNA-PK和pATM在phleomycin处理后的表达。与此同时,Mdig过表达细胞系对phleomycin所诱导的DNA双链断裂更敏感。相反的,用shRNA沉默ndig,可减缓DNA的双链断裂。结论：mdig通过与XRCC6的相互作用影响细胞DNA损伤的NHEJ修复通路,并导致phleomycin引起的DNA双链断裂更加敏感。
[Abstract]:Dust induced gene mdig (also known as mina53, MINA or N052) is associated with the increase of the expression of environmental induced gene.Mdig found in coal miners' macrophages and the incidence and prognosis of a variety of tumors. However, the effect of mdig on cell metabolism and tumor development remains to be elucidated. Studies have indicated that mdig proteins participate in the group through its JmjC domain. The demethylation of protein H3 in the glycine H3K9me3. However, unlike other classical demethylation enzymes, mdig lacks the protein domain that conjugates with chromatin. Thus the effect of ndig on chromatin or histone is more likely to be accomplished by interaction with other chromatin conjugproteins. The interaction between the dust induced gene protein and the intracellular functional proteins: This study aims to explore the functional proteins interacting with the dust induced gene ndig by proteomic analysis to explore the role of mdig in cell metabolism and tumor development. Square method: on human lung cancer cells A549 and human bronchus The antibody protein complex of mdig and IgG in the control group was precipitated by immunoprecipitation in the skin cell BEAS-2B. The protein complex was separated in SDS-PAGE gel, and the strip pattern of mdig protein was observed by coloring of komomesi blue. In the four groups of independent experiments, two different multi segment high performance liquid chromatography electrospray ionization tandem mass was used. Protein (Orbitrap Fusion and OrbitrapXL) analysis of proteins that are combined with mdig. The proteins screened were screened by immunoblotting and immunoblotting. Finally, the IPA software was used to analyze the molecular pathway of mdig interacting protein in A549 and BEAS-2B cells. Fruit: protein separation gel stained with Coomassie blue Three to five specific bands were stable in the protein samples of the mdig antibody precipitation and were not found in the control group IgG. The protein mass spectrometry analysis showed that some DNA repair related proteins and chromatin linked egg white could be detected in the protein complex of the mdig antibody precipitation, including XRCC5, XRCC6, RBBP4, CBX8, PRMT5, and TDRD. Western blot experiments further confirmed the binding of mdig to these proteins. In BEAS-2B cells, the interaction of mdig proteins is similar to that of A549 cells, but some proteins, such as CBX3 and CBX5, are observed only in BEAS-2B cells and interact with mdig. Conclusion: mdig through interaction with chaperone proteins is apparent. The functions of genetics, DNA repair, DNA replication and cell growth regulation. Second part of the dust induced gene protein is involved in the repair of DNA damage through the DNA non homologous terminal junction pathway: the repair of DNA double strand breaks (DSBs) is the key to maintain genomic integrity and prevent tumor formation in mammalian cells. Proteomics research This study aims to explore the role played by mdig in the NHEJ repair pathway and to verify the effect of mdig through the interaction with functional protein XRCC6, and the effect of mdig on the repair of DNA damage. Method: establishment of the effect of mdig in NHEJ repair pathway, and the effect of mdig on the repair of DNA damage. Methods: establishment of Mdig. Stable mdig overexpressed cell lines, mdig empty carrier infectious cell lines, mdig shRNA transfected gene silencing cell lines and control shRNA transfected cell lines. The changes of NHEJ pathway proteins in each cell line were measured by immunoblotting experiments with 30 gM humomycin (phleomycin), respectively. The expression of DNA fracture markers - gamma H2AX and pDNA-PKcs was observed by the immunofluorescence method. Comet assay detected the DNA damage sensitivity of each cell line to phleomycin treatment. Results: Mdig could interact with the non homologous terminal joint repair (NHEJ) protein XRCC6 interaction with DNA damage, and affect the key enzyme pDNA-PK of the NHEJ pathway and the table after the pATM. At the same time, Mdig overexpressed cell lines were more sensitive to the DNA double strand breaks induced by phleomycin. Conversely, the use of shRNA to silence ndig could slow the double strand breaks of DNA. Conclusion: mdig affects the NHEJ repair pathway of DNA damage in cells through the interaction with XRCC6, and leads to greater sensitivity of DNA double strand breaks caused by phleomycin.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R135.2

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