TGFβ信号通路下游分子Smad3和Smad4相互作用网络研究及功能初探

发布时间：2018-01-19 04:35

本文关键词： TGFβ 肝脏酵母双杂交蛋白相互作用　出处：《中国人民解放军军事医学科学院》2007年博士论文　论文类型：学位论文

【摘要】： TGFβ即转化生长因子(transforming growth factor)，，是一类多功能的细胞因子，其信号途径及相关功能也是近年来的研究热点。以往的研究表明，TGFβ与细胞的增殖、分化、迁移及凋亡有关；在机体中参与了从炎症反应、组织修复到骨骼形成、胚胎发育等一系列重要的生物学过程；并与某些人类疾病的发生密切相关。特别是在肝脏中，该信号通路的异常与肝炎、肝纤维化、肝硬化以及肝癌都有密切关系。因此对于该通路的研究具有十分重要的生物学意义；而对其各组分相互作用蛋白的研究将是阐明其功能及调控机制的关键所在。本文采用高通量规模化的蛋白质相互作用研究方法Y2H技术，以TGFβ信号通路中的重要分了Smad2、3、4为诱饵，在正常成人肝脏cDNA文库中进行相互作用蛋白的筛选。三个诱饵分子的筛选中，共获得125个阳性克隆。经过测序比对，排除非编码序列及不符合阅读框的序列后，得到二个诱饵的87个正确猎物克隆，匹配上32个基因共包含34个相互作用对。经文献调研，其中6对为已知的相互作用，剩余28对即为本研究新发现的相互作用。酵母回转验证中97％(33/34)的相互作用仍表现为阳性；随机选取10对相互作用进行CO-IP，阳性率达到了100％，表明相互作用的可信度较高。采用生物信息学的方法，结合基因敲除信息对得到的相互作用进行了生物学相关性分析。结果显示：66％的相互作用中猎物和诱饵具有相同的基因敲除表型；26％的猎物和诱饵具有基因共表达特性；26％的相互作用蛋白共享到GO中第六级功能注释；56％的相互作用从网络拓扑结构学分析具有较高的可信度。最后整合相关参数的评分结果，对每一对相互作用给出总体评分：53％(18/34)的相互作用具有高可信度。最后，将得到的相互作用以及整合了已知相互作用的数据进行可视化分析，构建相互作用网络图，对筛选结果进行补充和拓展。结合报告基因实验结果，我们推测筛选到的Smads相互作用蛋白在TGFβ信号通路中可能具有重要的调控作用。USP7：去泛素化酶，调控Smads蛋白在细胞中的表达水平；PPP1R12C：一种新的Smads蛋白去磷酸化途径，参与TGFβ信号的活性调节；SQSTM1：作为支架蛋白通过介导Smads与其它蛋白的相互作用发挥对该信号通路的调控作用；此外本文筛选到Smad4与NLK的相互作用，这提示Wnt/TGFβ信号通路可能具有新的交互联络方式。另外，本文对Smad3复合体的研究进行了初步探索，拟采用串联亲和纯化的方法在肝脏相关细胞系中分离纯化Smad3复合体进行质谱鉴定。首先构建了靶基因的逆转录病毒载体，转染包装细胞后进行了阳性克隆的筛选，结果待鉴定。综上所述，本文利用Y2H技术对TGF信号通路中的重要组分进行了相互作用蛋白的研究。通过其他方法的相互作用验证、生物信息学分析以及初步的功能实验初探，推测一些新筛选到的相互作用蛋白在TGFβ信号通路中可能具有重要的调控作用，为探索该通路发挥功能的新的作用机制提供了线索。
[Abstract]:TGF is the transforming growth factor beta (transforming growth factor), is a kind of multifunctional cytokine, its signal pathway and related functions is the hotspot in recent years. Previous studies showed that TGF and beta cell proliferation, differentiation, migration and apoptosis; in the body is involved in inflammation and tissue repair to bone formation, embryonic development and a series of important biological processes; and is closely related to the occurrence of certain human diseases. Especially in the liver, liver fibrosis and the abnormal signal pathway, hepatitis, cirrhosis and liver cancer are closely related. So it has very important biological significance for the study of the pathway of the study; the protein interactions in each group will be the key to clarify the function and regulatory mechanism.
This paper uses high-throughput scale protein interaction research methods of Y2H technology, the TGF beta signaling pathway plays an important role in the Smad2,3,4 as bait, screening interaction protein in normal human liver cDNA library. The three screening baits, 125 positive clones were obtained. After sequencing, the exclusion of non encoding sequence and does not meet the reading frame sequence, two bait 87 correct prey, cloning, the interaction of 34. Contains a total of 32 genes on the literature research, of which 6 of the known interaction, the remaining 28 of the interaction of the new findings.
In 97% yeast rotary verification (33/34) interaction is shown to be positive; the interaction of 10 randomly selected for CO-IP, the positive rate reached 100%, showed high reliability of the interaction. By using the method of bioinformatics, combined with gene knockout on the information interaction of biological correlation analysis. The results showed the prey and bait: interaction in 66% with the same gene knockout phenotype; 26% with characteristics of prey and bait gene co expression; interacting protein 26% share to sixth GO in 56% functional annotation; the interaction in the network topological analysis has higher reliability. Finally the integration parameters related to the scoring results for each of the interaction, presented the total score: 53% (18/34) interaction with high confidence.
Finally, the interaction and integration of the known interaction data visualization analysis, construct the interaction network diagram, supplement and extension of the screening results. Combined with the reporter gene assay results, we speculate that the screening of the proteins interacting with Smads may play an important role for.USP7 in regulation of TGF beta signaling pathway to ubiquitin enzyme, regulating the expression levels of Smads protein in cells; PPP1R12C: a new Smads protein phosphorylation pathway is involved in regulation of TGF beta signaling activity; SQSTM1 interaction as a scaffold protein and other proteins mediated by Smads play regulatory role in the signaling pathway; in addition to the screening interaction Smad4 and NLK, suggesting that Wnt/TGF beta signaling pathway may play a new interactive contact.
In addition, this paper makes a preliminary study on the Smad3 complex, the tandem affinity purification method for purification of Smad3 complex were identified in isolated liver related cells. First constructed retroviral vector of target gene, then transfected into packaging cell clones were screened, the results to be identified.
In summary, the important group in the TGF signaling pathway were studied interacting proteins by using Y2H technology. Through the interaction of other methods of verification, analysis and preliminary experimental study on the function of bioinformatics, presumably some novel interacting proteins screened may play an important role in the regulation of TGF beta signaling pathway. Provide clues for exploring the pathway to play the new mechanism 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】：2007
【分类号】：R363

【共引文献】