免疫共沉淀联合质谱筛选肝核因子HNF3β相互作用蛋白质及初步功能研究
发布时间：2018-11-17 07:12
【摘要】：肝细胞核因子HNF3β是一类在肝脏中富集表达的转录因子,调控多种肝脏基因的组织特异性表达,参与维持肝脏正常的表型与生理功能。敲除HNF3β的小鼠不能形成正常的神经脊索并早期胚胎致死, HNF3β的变异可能与2型糖尿病的发生相关。HNF3β参与糖代谢、脂肪代谢、调控胆酸以及生长相关基因的表达调控。HNF3β在调控基因转录的过程中需要募集多种辅助因子协同发挥作用。对HNF3β相互作用蛋白质的挖掘研究,有助于更深入的探索HNF3β在生命过程中的功能及作用机制。目前有关HNF3β蛋白质间相互作用的报道还相对较少,已知的与HNF3β发挥相互作用的蛋白质只有8个,包括OTX2,SRC1,TLE1,HOXA5,HNF6A,GSC,EN2,LHX1等[9-13],它们都参与转录调控。这些数据对于全面的阐明HNF3β蛋白复合体的组成及动态变化还是远远不够的。目前大规模研究蛋白质间相互作用的方法主要有酵母双杂交技术,但由于要去除转录激活区,因此并不适用于研究转录因子。免疫共沉淀技术可用于研究生理水平下蛋白质间的相互作用,随着质谱技术的发展,其准确性高与速度快的优点使免疫共沉淀联合质谱分析技术已被广泛应用于寻找生理水平下新的相互作用。 本研究对HNF3β相互作用蛋白质进行了分离鉴定,并对LMNA-HNF3β相互作用介导的生物学功能进行了初步的研究。以HNF3β特异抗体免疫沉淀分离HepG2细胞内HNF3β蛋白质复合体,SDS-PAGE电泳、考马斯亮蓝染色,切取与对照组存在差异的条带进行SDS-PAGE胶上蛋白质LTQ质谱鉴定其组成成分。本文通过4次独立重复实验共获得53个相互作用数据,生物信息学分析发现,其中与HNF3β共定位的蛋白质有9个,具有相互作用结构域的的蛋白质有5个,有10个分子同靶蛋白具有相同GO注释,有9个分子与靶蛋白之间存在间接相互作用。基于这些分析结果,挑选其中4个相互作用分子,构建真核表达载体,进行报告基因实验和外源性免疫共沉淀实验的验证。我们验证了LMNA与HNF3β的相互作用,报道基因实验表明LMNA可以影响HNF3β的转录活性。免疫共聚焦实验发现LMNA与HNF3β共定位于细胞核,SiLMNA下调HepG2细胞内LMNA的表达,可以改变HNF3β下游靶基因的表达。 基于这些实验可推测HNF3β可和细胞内多种蛋白质发挥作用,协同参与转录调控, LMNA-HNF3β之间相互作用的实验研究表明它们之间的相互作用可能具有重要的生理意义,并可能在葡萄糖、脂肪代谢及相关疾病的发生、发展中起作用。
[Abstract]:Liver nuclear factor HNF3 尾 is a kind of transcription factor which is enriched and expressed in the liver. It regulates the tissue specific expression of many liver genes and participates in maintaining the normal phenotype and physiological function of the liver. Mice knockout HNF3 尾 could not form normal neural chordal cord and die from early embryo. The variation of HNF3 尾 may be related to the development of type 2 diabetes mellitus. HNF3 尾 is involved in glucose metabolism and fat metabolism. In order to regulate the expression of cholic acid and growth-related genes, HNF3 尾 needs to recruit a variety of cofactors to play a synergistic role in the regulation of gene transcription. The research of HNF3 尾 interaction protein mining is helpful to further explore the function and mechanism of HNF3 尾 in the life process. At present, there are relatively few reports on the interaction between HNF3 尾 proteins. There are only 8 known proteins interacting with HNF3 尾, including OTX2,SRC1,TLE1,HOXA5,HNF6A,GSC,EN2,LHX1 [9-13], all of which are involved in transcriptional regulation. These data are not enough to fully elucidate the composition and dynamic changes of HNF3 尾-protein complex. At present, yeast two-hybrid technology is the main method to study protein interaction on a large scale, but it is not suitable for studying transcription factors because of removing the transcriptional activation region. Immune coprecipitation can be used to study protein interactions at physiological level, and with the development of mass spectrometry, Because of its high accuracy and high speed, immunoprecipitation combined with mass spectrometry has been widely used to search for new interactions at physiological level. In this study, HNF3 尾 interaction proteins were isolated and identified, and the biological function mediated by LMNA-HNF3 尾 interaction was studied. The HNF3 尾 protein complex in HepG2 cells was isolated by immunoprecipitation with HNF3 尾 specific antibody, SDS-PAGE electrophoresis, Coomassie brilliant blue staining, and the different bands were cut out and identified by LTQ mass spectrometry on SDS-PAGE gel. In this paper, 53 interaction data were obtained by 4 independent repeated experiments. Bioinformatics analysis showed that 9 proteins were colocated with HNF3 尾 and 5 proteins had interaction domains. Ten molecules have the same GO annotation as the target protein, and 9 have indirect interaction with the target protein. Based on these results, four of the interacting molecules were selected to construct the eukaryotic expression vector, and the reporter gene and exogenous co-immunoprecipitation experiments were carried out. We confirmed the interaction between LMNA and HNF3 尾, and reported that LMNA could affect the transcriptional activity of HNF3 尾. Confocal immunoassay showed that LMNA and HNF3 尾 co-located in the nucleus, and SiLMNA down-regulated the expression of LMNA in HepG2 cells, which could change the expression of HNF3 尾 downstream target gene. Based on these experiments, we can speculate that HNF3 尾 can play a role in many proteins and participate in the regulation of transcription. The experimental studies on the interaction between LMNA-HNF3 尾 and LMNA-HNF3 尾 suggest that the interaction between them may have important physiological significance. And may play a role in the development of glucose, fat metabolism and related diseases.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R341
本文编号：2336918
[Abstract]:Liver nuclear factor HNF3 尾 is a kind of transcription factor which is enriched and expressed in the liver. It regulates the tissue specific expression of many liver genes and participates in maintaining the normal phenotype and physiological function of the liver. Mice knockout HNF3 尾 could not form normal neural chordal cord and die from early embryo. The variation of HNF3 尾 may be related to the development of type 2 diabetes mellitus. HNF3 尾 is involved in glucose metabolism and fat metabolism. In order to regulate the expression of cholic acid and growth-related genes, HNF3 尾 needs to recruit a variety of cofactors to play a synergistic role in the regulation of gene transcription. The research of HNF3 尾 interaction protein mining is helpful to further explore the function and mechanism of HNF3 尾 in the life process. At present, there are relatively few reports on the interaction between HNF3 尾 proteins. There are only 8 known proteins interacting with HNF3 尾, including OTX2,SRC1,TLE1,HOXA5,HNF6A,GSC,EN2,LHX1 [9-13], all of which are involved in transcriptional regulation. These data are not enough to fully elucidate the composition and dynamic changes of HNF3 尾-protein complex. At present, yeast two-hybrid technology is the main method to study protein interaction on a large scale, but it is not suitable for studying transcription factors because of removing the transcriptional activation region. Immune coprecipitation can be used to study protein interactions at physiological level, and with the development of mass spectrometry, Because of its high accuracy and high speed, immunoprecipitation combined with mass spectrometry has been widely used to search for new interactions at physiological level. In this study, HNF3 尾 interaction proteins were isolated and identified, and the biological function mediated by LMNA-HNF3 尾 interaction was studied. The HNF3 尾 protein complex in HepG2 cells was isolated by immunoprecipitation with HNF3 尾 specific antibody, SDS-PAGE electrophoresis, Coomassie brilliant blue staining, and the different bands were cut out and identified by LTQ mass spectrometry on SDS-PAGE gel. In this paper, 53 interaction data were obtained by 4 independent repeated experiments. Bioinformatics analysis showed that 9 proteins were colocated with HNF3 尾 and 5 proteins had interaction domains. Ten molecules have the same GO annotation as the target protein, and 9 have indirect interaction with the target protein. Based on these results, four of the interacting molecules were selected to construct the eukaryotic expression vector, and the reporter gene and exogenous co-immunoprecipitation experiments were carried out. We confirmed the interaction between LMNA and HNF3 尾, and reported that LMNA could affect the transcriptional activity of HNF3 尾. Confocal immunoassay showed that LMNA and HNF3 尾 co-located in the nucleus, and SiLMNA down-regulated the expression of LMNA in HepG2 cells, which could change the expression of HNF3 尾 downstream target gene. Based on these experiments, we can speculate that HNF3 尾 can play a role in many proteins and participate in the regulation of transcription. The experimental studies on the interaction between LMNA-HNF3 尾 and LMNA-HNF3 尾 suggest that the interaction between them may have important physiological significance. And may play a role in the development of glucose, fat metabolism and related diseases.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R341
【共引文献】
相关期刊论文 前2条
1 陆明;刘丽梅;;翼螺旋转录因子Foxa2与糖脂代谢及糖尿病关系的研究进展[J];上海交通大学学报(医学版);2011年04期
2 孙婷婷;宋丽娜;于淼;李长燕;杨晓明;汪思应;;免疫共沉淀联合质谱对肝细胞核因子3β蛋白复合体的分离鉴定[J];生物技术通讯;2011年05期
，本文编号：2336918
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