PRMT5的转录调控及其泛素化-蛋白酶体降解机制研究

发布时间：2018-03-16 23:32

本文选题：PRMT5　切入点：NF-Y　出处：《暨南大学》2015年博士论文　论文类型：学位论文

【摘要】：PRMT5(Protein arginine methyltransferase 5),即蛋白精氨酸甲基化转移酶5,它属于II型甲基化转移酶,可以对称性地甲基化组蛋白或者非组蛋白底物的精氨酸残基,通过表观遗传的方式调控靶基因的表达或通过翻译后甲基化修饰途径调节关键的信号分子。最近的研究表明,PRMT5在许多人类肿瘤如肺癌、卵巢癌、结肠直肠癌、乳腺癌、黑色素瘤、白血病或淋巴瘤及恶性胶质瘤中呈高表达,且其高表达水平直接与肿瘤的进程及预后差密切相关。值得注意的是,敲低PRMT5可以抑制这些肿瘤细胞的增殖或诱导细胞凋亡,这些研究表明PRMT5可能是潜在的癌基因。然而,肿瘤中PRMT5的表达如何被调控在很大程度上并不清楚。因此,本研究旨在从转录及翻译后水平探讨肿瘤细胞中PRMT5高表达的调控机制。第一,我们从转录水平探讨肿瘤细胞中PRMT5高表达的调控机制。Hu实验室的前期观察表明,PRMT5在前列腺癌组织中呈高表达。因此,为了进一步研究PRMT5在前列腺癌中的转录激活机制,我们选择前列腺癌最常见的三个细胞系LNCa P,PC-3及DU 145细胞进行实验。首先证明在LNCaP细胞中PRMT5的启动子是杂合子,不同的等位基因(allele)之间具有不同的SNPs(Single Nucletide Polymorphisms)及13 bp Indel(Insertion/Deletion Polymorphisms)基因多态性。接着,我们利用双荧光素酶报告基因(Dual-Luciferase Reporter Gene)鉴定找到PRMT5的核心启动子位于-240 bp区域以内,并用EMSA(Electrophoresis Mobility Shift Assay)、ChIP(Chromatin Immunoprecipitation)、定点突变、删减突变等分子生物学方法从多个角度证明,在多个肿瘤细胞系中转录因子NF-Y(Nuclear Factor Y)结合到PRMT5核心启动子的CCAAT盒上进而调控PRMT5的转录激活。另外,我们发现下调PRMT5的表达所导致的细胞增殖抑制直接与NF-YA的敲低有关。更重要的是,研究结果证明PKC-c-Fos信号通路可以通过下调NF-YA的表达而抑制PRMT5的转录。鉴于多个PKC(Protein Kinase C)家族的成员被报道在某些肿瘤中表达水平下调,我们推测PKC-c-Fos信号通路的失活可能与某些肿瘤病人中PRMT5的高表达有关,而深入研究PRMT5如何与PKC/c-Fos信号通路的相互交叉将为进一步阐释PRMT5促肿瘤发生的角色提供理论依据。第二,我们从翻译后修饰水平探讨PRMT5蛋白表达的调控机制。基于上面的结果及我们未发表的发现,我们推测PRMT5自身可以被翻译后修饰。为此,使用Co-IP(Co-Immunoprecipitation)及WB(Western blot)技术证实PRMT5蛋白可以发生多聚泛素化翻译后修饰。通过Co-IP及定点突变,证实PRMT5上的多个赖氨酸位点对于PRMT5的多聚泛素化修饰均很重要。接着,使用MS(Mass Spectrometry)、Co-IP、GST下拉、亚细胞共定位、BiFC、定点突变、删减突变等分子生物学方法,我们找到并证明CHIP(Carboxy terminus of Heat shock cognate 70-Interacting Protein)是与PRMT5相互作用的E3连接酶,后者可以与分子伴侣(chaperones)系统如热休克蛋白90及70(Hsp90/Hsp70)发生相互作用,进而通过泛素化依赖的蛋白酶体降解方式调控PRMT5/MEP50(Methylosome protein 50)的表达。另外,我们的结果显示Hsp90的抑制剂如GA(Geldanamycin)或17-AAG(17-(Allylamino)-17-demethoxygeldanamycin)介导的PRMT5下调依赖CHIP E3连接酶介导的蛋白酶体降解机制,且17-AAG可以协同过表达CHIP对PRMT5表达的抑制作用及诱导细胞凋亡的效应。这些发现提示在肿瘤组织中CHIP表达水平的下调可能与PRMT5的高表达水平相关。最后,我们证明靶向PRMT5联合17-AAG干预可作为一个新的方法抑制肿瘤细胞的生长。综上所述,我们探讨了PRMT5的转录及翻译后修饰的调控机制。研究结果表明,转录因子NF-Y对激活PRMT5的转录很关键,且NF-Y的转录活性受到PKC/c-Fos信号通路的负调控。另一方面,E3连接酶CHIP与分子伴侣系统相偶联,介导PRMT5翻译后的泛素化修饰及蛋白酶体依赖的降解。这些研究将为促进靶向PRMT5及其上游相关信号分子的抗癌治疗提供理论和实验依据,为新抗癌药物的研发奠定基础。
[Abstract]:PRMT5 (Protein arginine methyltransferase 5), namely protein arginine methyltransferase 5, which belongs to the type II methyltransferase, can arginine residues symmetrically methylated histone and non histone substrates, regulation of the key signaling molecule through epigenetic regulation of target gene expression of or through posttranslational methylation pathways. Recent studies show that PRMT5 in many human cancers such as lung cancer, ovarian cancer, colorectal cancer, breast cancer, melanoma, leukemia or lymphoma and malignant glioma showed high expression, and its high expression level directly with tumor progression and poor prognosis are closely related. It is worth noting that knockdown of PRMT5 inhibited tumor cell proliferation or apoptosis, these studies suggest that PRMT5 may be a potential oncogene. However, how to express the tumor in the PRMT5 is regulated to a great extent Is not clear. Therefore, this study aims to explore the regulatory mechanism of PRMT5 expression in tumor cells from the transcription and translation level. First, we investigate the early laboratory observation at the level of transcription regulatory mechanism of.Hu high expression of PRMT5 in tumor cells showed that the expression of PRMT5 in prostate cancer tissues was. Therefore, in order to further transcription the research of PRMT5 in prostate cancer activation mechanism, we choose the most common prostate cancer cell line LNCa three P, PC-3 and DU 145 cells were used. In the first show that the PRMT5 promoter in LNCaP cells is heterozygous for different alleles (allele) with different SNPs (Single Nucletide Polymorphisms) 13 BP and Indel (Insertion/Deletion Polymorphisms) gene polymorphism. Then, we use dual luciferase reporter gene (Dual-Luciferase Reporter Gene) to find the core promoter of PRMT5 identification The promoter is located within -240 BP region, and EMSA (Electrophoresis Mobility Shift Assay), ChIP (Chromatin Immunoprecipitation), point mutation, deletion mutation and other molecular biology methods proved from many angles, in a number of transcription factors in cancer cell lines NF-Y (Nuclear Factor Y) binding to the PRMT5 promoter, CCAAT box transcriptional activation of PRMT5. In addition, we found that the down-regulation of PRMT5 expression caused by the inhibition of cell proliferation directly with NF-YA knockdown. More importantly, the results demonstrated that the transcription of PKC-c-Fos signaling pathway can inhibit PRMT5 by down regulating the expression of NF-YA and in view of multiple PKC (Protein Kinase C) family members the reported level of down regulated expression in some tumors, we hypothesized that the activity may be related to the high expression of PRMT5 in some tumor patients in the PKC-c-Fos signaling pathway, and in-depth study of how PRMT5 And cross the PKC/c-Fos signaling pathway will further explain the role PRMT5 promoting tumorigenesis and provide a theoretical basis. From second, we discuss the regulation mechanism of the level of post-translational modification of PRMT5 protein expression. Based on the above results, we found unpublished, we speculate that PRMT5 itself can be modified after translation. Therefore, the use of Co-IP (Co-Immunoprecipitation) and WB (Western blot) technology confirmed that PRMT5 protein could be polyubiquitinated post-translational modification. Through Co-IP and PRMT5 point mutations, confirmed multiple lysine sites for PRMT5 poly ubiquitination is very important. Then, using the MS (Mass Spectrometry), Co-IP, GST pull-down, subcellular co localization, BiFC, point mutation, deletion mutation and other molecular biological methods, we find that CHIP (Carboxy terminus of Heat shock cognate 70-Interacting Protein) is PRMT5 The interaction of E3 ligase, which can be used with molecular chaperone (chaperones) systems such as heat shock protein 90 and 70 (Hsp90/Hsp70) interact with proteasome degradation through the ubiquitin dependent regulation of PRMT5/MEP50 (Methylosome protein 50) expression. In addition, our results show that GA inhibitors such as Hsp90 (Geldanamycin) or 17-AAG (17- (Allylamino) -17-demethoxygeldanamycin) mediated down-regulation of PRMT5 dependent proteasomal degradation mechanism of CHIP E3 ligase mediated, and 17-AAG can be co overexpression of CHIP on the expression of PRMT5 and inhibition effect by inducing apoptosis. These findings suggest that the expression of CHIP in tumor tissue is related to the down-regulation of PRMT5 and high the expression level. Finally, we show that targeting PRMT5 and 17-AAG intervention can be used as a new method to suppress the growth of tumor cells. To sum up, we discussed PR The regulation mechanism of modification of transcription and translation of MT5. The results show that the transcription of NF-Y on activation of PRMT5 is the key transcription activity and NF-Y negative regulation by PKC/c-Fos signaling pathway. On the other hand, coupled with E3 ligase CHIP and molecular chaperone systems, mediated ubiquitination and proteasomal degradation of PRMT5 translation after the dependent. These studies will provide theoretical and experimental basis for the promotion of anticancer therapy targeting PRMT5 and its upstream signal molecule, lay the foundation for the development of new anticancer drugs.

【学位授予单位】：暨南大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R730.2

【共引文献】