串联亲和纯化技术联合质谱鉴定RSK4及其变异体的相互作用蛋白

发布时间：2018-06-18 01:21

本文选题：乳腺癌 + RSK4　；参考：《广西医科大学》2015年硕士论文

【摘要】：研究背景：p90核糖体S6蛋白激酶4(Ribosomal S6 protein kinase 4, RSK4)是一新发现的抑制乳腺癌侵袭转移的重要因子,它能抑制乳腺癌细胞的生长并诱导其衰老；而RSK4前体mRNA在可变剪切作用下形成可变剪接的变异体(RSK4m),并具有不同的生物学功能。迄今为止,RSK4及其变异体RSK4m在乳腺癌中作用机制所知甚少。蛋白质是生命活动的主要物质,蛋白质功能的实现,离不开与其它蛋白质之间的相互作用。可变剪接是调节基因表达和产生蛋白质多样性的重要机制。前期研究我们认为RSK4及其变异体与其它蛋白通过相互作用形成不同的蛋白质复合物后在乳腺癌发生发展中发挥重要的作用。本研究拟建立哺乳动物体系中的串联亲和纯化技术,并联合质谱分析鉴定RSK4及其变异体相互作用蛋白质,经生物信息学方法分析找寻RSK4及其变异体相互作用蛋白中可能存在的关键蛋白,初步验证其生物学功能。本研究通过对RSK4和其变异体RSK4m的相互作用蛋白的研究,可以为我们初步了解其独特的蛋白功能和作用机制,从而为进一步阐明乳腺癌发生发展机制及寻找乳腺癌治疗新靶点开辟新思路和新途径。研究方法：分别构建了带有串联亲和标签(两个strep II标签和一个FLAG标签)的RSK4和RSK4m过表达载体。转染到RSK4低表达的乳腺癌细胞株MDA-MB-231中,运用串联亲和纯化技术(tandem affinity purification, TAP),得到与RSK4或RSK4m相互作用的蛋白复合物,然后对所得的蛋白复合物进行质谱鉴定和分析,应用生物信息学分析方法(GO和IPA分析),分别对RSK4和RSK4m的相互作用蛋白进行功能注释和绘制了蛋白-蛋白相互作用网络图。为了确认生物信息学分析结果,我们还利用免疫共沉淀(Co-Immunoprecipitation, CO-IP)对其中PRMT5(蛋白精氨酸甲基转移酶5)蛋白与RSK4的相互作用关系进行验证。研究结果：本研究通过两个Strep II标签和单个FLAG标签为基础的串联亲和纯化技术(SF-TAP)联合运用nano LC-MS/MS质谱技术分别分离和鉴定出82个RSK4相关的相互作用蛋白,137个RSK4m相关的相互作用蛋白。应用生物信息学分析方法(GO和IPA分析),分别对RSK4和RSK4m的相互作用蛋白进行功能注释和绘制了蛋白-蛋白相互作用网络图。GO(Gene Ontology)分析结果：RSK4 wt或RSK4m的互作蛋白参与细胞的多种生理活动,比如：tRNA代谢(tRNA metabolic process),核糖体蛋白复合物的生物合成(ribonucleoprotein complex biogenesis),氨基酸的活化(amino acid activation)和蛋白折叠(protein folding)等,这些互作蛋白绝大部分都位于细胞质中,与RSK4本身位于细胞质,在细胞质发挥生物功能是一致的。IPA (Ingenuity Pathway Analysis)分析结果：将鉴定到的互作蛋白经过核心数据分析方法(Core Analyze dateset),分别得到RSK4 wt和RSK4m各自三个富集度最高的信号通路和蛋白互作网络：①RSK4 wt和RSK4m主要参与了核糖体生物合成,互作蛋白组网络中的PRMT5、STAT3、 HSP90AB1、ATP5B、RPL核糖体亚基系列蛋白等与细胞中核糖体生物合成密切相关；②RSK4 wt和RSK4m的互作蛋白均参与细胞前体RNA的处理,互作蛋白中的HNRNPH1、HNRNPK、HNRNPM属于hnRNPs(heterogeneous nuclear ribonucleproteins)家族,是RNA结合蛋白,能与mRNA前体相互作用,影响mRNA前体的处理以及mRNA代谢和运输；③RSK4 wt和RSK4m主要参与染色体的重组与代谢,相互作用蛋白STK38、STK38L、 MOB2和DDX3X参与调节中心体的复制和有丝分裂期染色质的重组。综合GO分析和IPA分析的结果,表明RSK4 wt和RSK4m参与了多种细胞活动,拥有多种不同的生物学功能。RSK4m的相互作用蛋白更多,并且参与更多的生物通路。同时,CO-IP证实了其中的蛋白如PRMT5与RSK4存在相互作用关系,表明PRMT5可能可以通过甲基化RSK4从而抑制RSK4对细胞生长的抑制作用。研究结论：RSK4和RSK4m存在与其相互作用蛋白,相互作用蛋白复合物主要参与了核糖体生物合成、前体RNA处理和染色体的重组与代谢,并介导多种生物学功能调控,特别是有关细胞衰老的调控；而且RSK4和RSK4m有着不同相互作用蛋白,其调控作用及机制有可能不同,RSK4m可能比RSK4拥有更多的分子功能,但其中的具体功能尚待于进一步研究。而其中的互作蛋白PRMT5与RSK4存在直接的相互作用关系。
[Abstract]:Background: P90 ribosome S6 protein kinase 4 (Ribosomal S6 protein kinase 4, RSK4) is an important factor in inhibiting the invasion and metastasis of breast cancer, which inhibits the growth and senescence of breast cancer cells, and RSK4 precursor mRNA forms a variant spliced variant (RSK4m) under variable shear action, and has a different birth rate. Physical function. So far, RSK4 and its variant RSK4m have little knowledge of the mechanism of action in breast cancer. Protein is the main substance of life activity. The realization of protein function can not be separated from the interaction with other proteins. Variable splicing is an important mechanism for regulating gene expression and producing egg white diversity. It is considered that RSK4 and its variants and other proteins play an important role in the development of breast cancer after interacting with other proteins by interacting with other proteins. This study aims to establish a series affinity purification technology in mammalian system, and to identify the interaction proteins of RSK4 and its variants by mass spectrometry analysis. The possible biological function of RSK4 and its variant protein was found by method analysis. The study on the interaction protein of RSK4 and its variant RSK4m could provide us a preliminary understanding of its unique protein function and mechanism, so as to further elucidate the development of breast cancer. Mechanism and new approaches for finding new targets for breast cancer treatment. Research methods: RSK4 and RSK4m overexpression vectors with tandem affinity labels (two strep II tags and one FLAG tag) were constructed respectively. The transfected to RSK4 low expression breast cancer cell line MDA-MB-231, using serial affinity purification technology (tandem affinity PU). Rification, TAP), the protein complexes interacting with RSK4 or RSK4m were obtained, and then the protein complexes were identified and analyzed by mass spectrometry. The interaction proteins of RSK4 and RSK4m were explained by bioinformatics analysis method (GO and IPA analysis), and the protein protein interaction network diagram was plotted and plotted for the interaction proteins of RSK4 and RSK4m. As a result of bioinformatics analysis, we also used Co-Immunoprecipitation (CO-IP) to verify the interaction between PRMT5 (protein arginine methyltransferase 5) protein and RSK4. The results of this study were based on the tandem affinity purification technology (SF-TAP) based on two Strep II tags and single FLAG tags. 82 RSK4 related interacting proteins and 137 RSK4m related interacting proteins were isolated and identified by nano LC-MS/MS mass spectrometry. The interaction proteins of RSK4 and RSK4m were annotated and the protein protein interaction network diagram.GO (Gene) was plotted by bioinformatics analysis method (GO and IPA analysis). Ontology) analysis results: the interaction proteins of RSK4 WT or RSK4m participate in various physiological activities of the cells, such as the tRNA metabolism (tRNA metabolic process), the biosynthesis of the ribosomal protein complex (ribonucleoprotein complex biogenesis), the activation of the amino acids (amino) and protein folding, and so on. Most of the proteins are located in the cytoplasm, and RSK4 itself is located in the cytoplasm, and the.IPA (Ingenuity Pathway Analysis) analysis is consistent with the biological function of the cytoplasm. The identified mutual protein, through the core data analysis method (Core Analyze dateset), obtains the three highest concentration of RSK4 WT and RSK4m respectively. Signal pathway and protein interaction network: (1) RSK4 WT and RSK4m are mainly involved in ribosome biosynthesis, PRMT5, STAT3, HSP90AB1, ATP5B, RPL ribosome series proteins are closely related to the biosynthesis of ribosome biosynthesis in the cell network, and both RSK4 WT and RSK4m interaction proteins are involved in the treatment of cell precursor RNA. HNRNPH1, HNRNPK, and HNRNPM belong to the hnRNPs (heterogeneous nuclear ribonucleproteins) family, which are RNA binding proteins that interact with the mRNA precursors, affect the processing of mRNA precursors and mRNA metabolism and transport. To regulate the replication of the centrosome and the recombination of chromatin during mitosis. Combined with the results of GO analysis and IPA analysis, it shows that RSK4 WT and RSK4m are involved in a variety of cell activities, with a variety of different biological functions,.RSK4m interaction proteins, and more biological pathways. Meanwhile, CO-IP has confirmed the proteins such as PRMT5. The interaction with RSK4 indicates that PRMT5 may be able to inhibit the inhibitory effect of RSK4 on cell growth by methylation of RSK4. Conclusions: RSK4 and RSK4m exist with their interacting proteins. The interaction protein complex is mainly involved in ribosome biosynthesis, precursor RNA treatment and chromosome recombination and metabolism, and mediates A variety of biological functions and regulation, especially the regulation of cell senescence, and RSK4 and RSK4m have different interacting proteins, and their regulatory roles and mechanisms may be different. RSK4m may have more molecular functions than RSK4, but the specific functions of which are yet to be further studied. The mutual protein PRMT5 and RSK4 exist directly. The interaction between them.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R737.9

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