多重组学手段研究蛋白UHRF2的生物学功能及其机制

发布时间：2018-04-16 09:33

本文选题：UHRF2 + 液相串联质谱　；参考：《中国人民解放军军事医学科学院》2016年博士论文

【摘要】：UHRF家族蛋白是以具有多个结构域为特点的一组蛋白,包含4个成员。其中,以UHRF1研究最多,UHRF1被报道在多个生物学过程中发挥重要作用,比如它能够维持DNMT1介导的DNA甲基化。UHRF2具有与UHRF1相似的序列和结构域。这种结构上的相似性预示着两个蛋白之间功能的保守性。如同UHRF1,UHRF2也能够识别半甲基化DNA以及甲基化的组蛋白,在体外也被证明与DNMTs以及组蛋白甲基转移酶G9a相互作用。然而,UHRF2与UHRF1也有着显著的不同。UHRF2不能够挽救Uhrf1缺失胚胎干细胞的DNA甲基化缺陷,因为UHRF2没有在细胞周期S期招募DNMT1至复制位点的能力。此外,由于UHRF2-SRA结构域对羟基化的结合偏好性,UHRF2被报道是羟甲基化DNA的特异性结合因子。如同UHRF1,UHRF2同样被报道与肿瘤相关。但是对于UHRF2在肿瘤中表现的相关报道都比较浅显且模糊。一些研究称UHRF2表现出肿瘤抑制因子的特性,因为它能够干扰细胞周期的进程。而也有报道显示,由于UHRF2的表达量在某些肿瘤中上调,UHRF2可能具有潜在的原癌基因特性。关于UHRF2与肿瘤相关性的报道目前来说十分有限,UHRF2在肿瘤发生过程中的精确生物学功能,以及它是否如同UHRF1一样可能参与转移相关的过程,还需要进一步的研究。转移是恶性肿瘤的重要特点,90%的癌症死亡都是由于转移导致。癌症细胞的转移是一个非常复杂的过程,这个过程中,细胞发生上皮-间质转化,从而获得侵袭和转移的能力。在上皮-间质转化的过程中,上皮细胞失去细胞-细胞间连接和细胞极性,获得间质样细胞的特性,同时转移和侵袭能力增强。上皮-间质转化是一个多级调控的过程,转录因子激活调节下游目标蛋白的表达变化,从而扰动细胞-细胞间连接、细胞极性、骨架结构以及胞外基质降解等多个生物学过程。虽然有关上皮-间质转化的研究近年有许多报道,但相关转录因子如何发挥调控机制依然需要进一步的探索。蛋白质组学技术是生物学研究中进行大规模蛋白质分析的强大手段。本实验室近年建立了一套快速非标定量的流程,能够在12小时的质谱时间里鉴定8000个蛋白质。这使得我们可以快速地进行大量样本的鉴定。同时,我们还发明了一种转录因子富集技术,能够有效富集内源性转录因子这种生物体中的低丰度蛋白,并进行定量。将蛋白表达水平的检测与转录因子-DNA结合活性的检测相结合,使得我们能够关联转录因子的活性及其目标蛋白的表达量变化。来自转录因子及蛋白表达水平的双重信息的综合分析,能够为蛋白可能发挥的生物学功能以及参与的信号转导通路,提供重要的生物学线索。使用多重组学方式探索蛋白功能及机制,将是具有重要意义的揭示蛋白秘密的手段。在本篇文章中,为了探索UHRF2的功能,我们使用了蛋白表达水平的质谱鉴定,转录因子活性变化的质谱鉴定,IP-质谱鉴定以及Ch IP-seq测序等多维度组学手段,检测UHRF2在癌症细胞系中过表达时引起的生物学变化。首先我们检测了UHRF2在胃癌细胞系MKN74中过表达诱导的蛋白水平变化。在肽段1%FDR水平,共有7662个特异蛋白被质谱检测。经过统计学分析,192个蛋白呈现上调变化,297个蛋白表现出下调变化。进行GO分析后发现,上调蛋白主要定位于细胞核、大分子复合物及胞质内,而下调的蛋白质则主要位于细胞-细胞间连接、基底膜以及内膜系统中。鉴定结果显示UHRF2抑制了许多上皮细胞表面标志物的表达,包括CDH1、JUP、TJP1、DSG2、INADL、SPINT2、CXADR、SPINT1以及TJP2;同时,稳定了一些包括p53在内的重要核蛋白的表达。UHRF2过表达诱导的差异蛋白分析表明,UHRF2与细胞粘附及肿瘤转移有重要相关性。为了进一步探索UHRF2过表达诱导蛋白水平变化的驱动因素,我们使用了TFRE富集加质谱检测的手段,检测转录因子-DNA结合活性的变化。这使得我们能够观测到一组全蛋白鉴定无法检测的低丰度转录因子。在肽段1%FDR水平,我们检测到581个转录因子以及537个转录共调控因子。经过统计学分析,其中17个转录因子及8个转录共调控因子因UHRF2过表达而出现DNA结合活性的增强。根据文献报道,其中5个活性增强的转录因子被注释为上皮-间质转化相关转录因子。这些分析表明转录因子-DNA结合活性的变化与蛋白表达水平的变化具有非常好的一致性,且都支持同一个模型:UHRF2过表达导致EMT转录因子的激活以及EMT相关蛋白的表达变化。结合组学鉴定提供的信息,为了进一步验证UHRF2参与EMT过程,我们使用CRISPR-Cas9技术在恶性胃癌细胞系SGC7901以及BGC823中使用不同的引导RNA(g RNA)敲除UHRF2并进行Transwell实验评估细胞转移能力。与对照组相比,UHRF2敲除非常明显地抑制了细胞迁移和侵袭能力。此外我们还发现,UHRF2的缺失诱导SGC7901细胞发生了明显的形态变化,由间质样细胞向上皮样细胞转变,同时细胞之间发生聚集。这些结果表明UHRF2对调控细胞转移能力具有重要作用。我们使用UHRF2抗体及对照Ig G抗体进行了Ch IP实验以寻找UHRF2与DNA结合的位点,结果表明,UHRF2与CDH1的启动子区域有结合,可能作为一个转录共调控因子与EMT-TFs一起调节CDH1的表达。为了进一步证实这个猜想,我们进行了IP-质谱鉴定实验,以探寻UHRF2相互作用蛋白。结果表明,UHRF2与一些表观调控复合体相结合,且与EMT转录因子TCF7L2有相互作用,进一步提供了UHRF2参与EMT过程的证据。综上所诉,在本篇文章中,我们证明了UHRF2作为一个转录共调控因子通过调节上皮-间质转化过程影响肿瘤细胞的迁移和侵袭能力。我们在胃癌细胞系中过表达UHRF2蛋白,然后进行多维度的组学分析。全蛋白表达的鉴定结果提示UHRF2对细胞-细胞间连接具有抑制作用;转录因子-DNA结合活性的检测表明UHRF2过表达引起EMT转录因子活性的升高;生物学实验证明敲除UHRF2能够抑制肿瘤细胞的转移和侵袭能力;Ch IP-seq及相互作用蛋白组表明UHRF2作为一个转录共调控因子,与EMT转录因子一同参与EMT过程的调节。总之,我们的研究揭示了蛋白UHRF2的生物学功能,同时为蛋白功能研究提供了强大而有效的新手段,即通过多重组学鉴定,综合多个生物学层面的信息,获得蛋白功能及相关信号通路的全面线索,进而进行生物学功能的验证。
[Abstract]:UHRF is a protein family with multiple domains for a group of protein characteristics, contains 4 members. Among them, the most studied UHRF1, UHRF1 was reported to play an important role in many biological processes, such as it can maintain DNA methylation of.UHRF2 DNMT1 mediated with sequence and domain structure similar to that of UHRF1. This structural similarity indicates the function between the two protein conserved. Like UHRF1, UHRF2 is also able to identify the hemi methylated DNA and methylated histones, in vitro was also demonstrated with DNMTs and histone methyltransferase G9a interaction. However, UHRF2 and UHRF1 have different.UHRF2 is not significant can save the Uhrf1 deletion of DNA methylation defects in embryonic stem cells, because UHRF2 is not in the S phase of the cell cycle to recruit DNMT1 replication sites ability. In addition, due to the UHRF2-SRA domain of the hydroxylated binding preference of UHRF2 was reported Is the specificity of hydroxymethylated DNA binding factor. Like UHRF1, UHRF2 is also reported associated with cancer. But for the reported expression of UHRF2 in tumors are relatively simple and fuzzy. Some studies showed UHRF2 characteristics of tumor suppressor, because it can interfere with cell cycle progression and also. Reported that, due to the expression of UHRF2 in some tumors increased, UHRF2 may have potential. The characteristics of the original cancer gene on UHRF2 and tumor related reports at present is very limited, UHRF2 in tumor biology process can work accurately, and whether it is like UHRF1 may be involved in processes related to the transfer, also need further study transfer is an important feature of malignant tumors, 90% of cancer deaths are due to metastasis result. Metastasis of cancer cells is a very complex process, the process of cell Epithelial mesenchymal transition, resulting in the invasion and metastasis ability. In epithelial mesenchymal transition process, epithelial cells lose cell-cell junctions and cell polarity, obtain the characteristics of mesenchymal cells, the metastasis and invasion ability. Epithelial mesenchymal transition is a process control the transcription factor activator expression regulate downstream target proteins, thereby perturbing cell-cell junctions, cell polarity, skeleton structure and extracellular matrix degradation in many biological processes. Although the epithelial mesenchymal transition research in recent years there are many reports, but the transcription factors related to how to play the regulation mechanism still needs further exploration. Proteomics is a powerful technique for protein analysis by means of large-scale biological research laboratory in recent years. This has established a fast non quantitative process, can be in 12 hours of mass spectrometry Time in the identification of 8000 proteins. This allows us to quickly identify a large number of samples. At the same time, we also developed a transcription factor enrichment technology, can effectively enrich low abundance proteins of endogenous transcription factor in the organism, and quantitatively. The protein expression level of -DNA and transcription factor binding activity detection the combination, so that we can change the expression of target protein activity and associated transcription factors. Comprehensive analysis from the transcription factor and protein expression level of dual information, to biological functions of proteins may play and participate in the signal transduction pathway, provide important clues to the biology. Using recombinant protein function and mechanism of science exploration that would be of great significance to reveal the protein secret means. In this article, in order to explore the function of UHRF2, we make use of the The expression level of protein identification by mass spectrometry, mass spectrometry identification of transcription factor activity, IP- and Ch IP-seq sequencing identified multi-dimensional group by means of biological changes caused by detecting the expression of UHRF2 in cancer cell lines. At first we examined the UHRF2 in gastric cancer cell line MKN74 in protein expression level changes induced in. Peptide 1%FDR levels, a total of 7662 specific protein by mass spectrometry. After statistical analysis, 192 proteins showed an increasing change, 297 proteins exhibited down-regulation changes. GO analysis found that the up-regulated protein mainly localized in the nucleus, macromolecular complexes and the cytoplasm, and down regulated proteins are mainly located in the cell - the connections between cells, basement membrane and membrane system. The identification results show that UHRF2 inhibits many epithelial cell surface marker expression, including CDH1, JUP, TJP1, DSG2, INADL, SPINT2, CXADR, SPINT1 At the same time, stability and TJP2; the expression of.UHRF2 and some important nuclear proteins including p53 protein induced analysis showed that overexpression of UHRF2 with cell adhesion and tumor metastasis have important correlation. In order to further explore the expression of UHRF2 protein level changes induced by driving factors, we use the TFRE enrichment plus mass spectrometry method. The changes of the activity of transcription factor -DNA. Combined detection of low abundance transcription factor which allows us to observe a whole group of protein identification cannot be detected. At the peptide level of 1%FDR, we detected 581 transcription factors and 537 transcriptional co regulation factor. Through statistical analysis, of which 17 transcription factors and 8 transcriptional co regulation the enhancement factor by UHRF2 overexpression and DNA binding activity. According to the literatures, the transcription factor activity was enhanced 5 notes for the EMT related transcription factors . these analyses show that the transcription factor -DNA binding protein activity change and expression has very good consistency, and will support the same model: UHRF2 leads to activation of the EMT transcription factors and the expression of EMT related protein expression changes. Combined with the group identification information provided, in order to further verify the UHRF2 in EMT the process, use the guide of different RNA we use CRISPR-Cas9 SGC7901 in malignant gastric cancer cell lines and BGC823 (g RNA) and UHRF2 knockout Transwell experimental assessment of metastasis ability. Compared with the control group, UHRF2 knockout very significant inhibition of cell migration and invasion. In addition, we also found that the occurrence of morphological changes obviously SGC7901 cells induced by UHRF2 deletion, change from mesenchymal cells into epithelioid cells, and between cells aggregation. These results suggest that the regulation of UHRF2 on cells Has an important role in metastasis. We use the UHRF2 antibody and G antibody control Ig Ch IP experiment to find the combination of UHRF2 and DNA loci, the results show that the promoter region of UHRF2 and CDH1 combination, expression may act as a transcriptional co regulation factor together with EMT-TFs regulation CDH1. To further confirm this conjecture. We conducted IP- mass spectrometry experiments, to explore the interaction protein of UHRF2 and UHRF2. The results show that some epigenetic regulation of complex combination and interaction with EMT transcription factor TCF7L2, provides further evidence of UHRF2 involved in the EMT process. To sum up, in this article, we show that UHRF2 as a a total of transcription regulatory factor by regulating epithelial mesenchymal transition process affecting tumor cell migration and invasion. The overexpression of UHRF2 protein in gastric cancer cell lines, and then multi dimension Proteomic analysis of total protein expression. The identification results showed that UHRF2 of cell-cell connection has inhibitory effect; transcription factor -DNA binding activity assay showed that UHRF2 overexpression increased EMT transcription factor activity; biological experiments show that knockdown of UHRF2 can inhibit the invasion and metastasis of tumor cells; Ch and IP-seq interacting protein group show UHRF2 as a transcriptional co regulation factor regulating transcription factor EMT to participate in the EMT process. In conclusion, our study reveals the biological function of UHRF2 protein, while the protein function research provides a powerful and effective, that is through the reorganization and identification, multi level biological information obtained comprehensive clues protein function and signal pathway, and then verify its biological function.

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q51

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