免疫共沉淀聯(lián)合質(zhì)譜篩選肝核因子HNF3β相互作用蛋白質(zhì)及初步功能研究
發(fā)布時間：2018-11-17 07:12
【摘要】：肝細(xì)胞核因子HNF3β是一類在肝臟中富集表達(dá)的轉(zhuǎn)錄因子,調(diào)控多種肝臟基因的組織特異性表達(dá),參與維持肝臟正常的表型與生理功能。敲除HNF3β的小鼠不能形成正常的神經(jīng)脊索并早期胚胎致死, HNF3β的變異可能與2型糖尿病的發(fā)生相關(guān)。HNF3β參與糖代謝、脂肪代謝、調(diào)控膽酸以及生長相關(guān)基因的表達(dá)調(diào)控。HNF3β在調(diào)控基因轉(zhuǎn)錄的過程中需要募集多種輔助因子協(xié)同發(fā)揮作用。對HNF3β相互作用蛋白質(zhì)的挖掘研究,有助于更深入的探索HNF3β在生命過程中的功能及作用機(jī)制。目前有關(guān)HNF3β蛋白質(zhì)間相互作用的報(bào)道還相對較少,已知的與HNF3β發(fā)揮相互作用的蛋白質(zhì)只有8個,包括OTX2,SRC1,TLE1,HOXA5,HNF6A,GSC,EN2,LHX1等[9-13],它們都參與轉(zhuǎn)錄調(diào)控。這些數(shù)據(jù)對于全面的闡明HNF3β蛋白復(fù)合體的組成及動態(tài)變化還是遠(yuǎn)遠(yuǎn)不夠的。目前大規(guī)模研究蛋白質(zhì)間相互作用的方法主要有酵母雙雜交技術(shù),但由于要去除轉(zhuǎn)錄激活區(qū),因此并不適用于研究轉(zhuǎn)錄因子。免疫共沉淀技術(shù)可用于研究生理水平下蛋白質(zhì)間的相互作用,隨著質(zhì)譜技術(shù)的發(fā)展,其準(zhǔn)確性高與速度快的優(yōu)點(diǎn)使免疫共沉淀聯(lián)合質(zhì)譜分析技術(shù)已被廣泛應(yīng)用于尋找生理水平下新的相互作用。 本研究對HNF3β相互作用蛋白質(zhì)進(jìn)行了分離鑒定,并對LMNA-HNF3β相互作用介導(dǎo)的生物學(xué)功能進(jìn)行了初步的研究。以HNF3β特異抗體免疫沉淀分離HepG2細(xì)胞內(nèi)HNF3β蛋白質(zhì)復(fù)合體,SDS-PAGE電泳、考馬斯亮藍(lán)染色,切取與對照組存在差異的條帶進(jìn)行SDS-PAGE膠上蛋白質(zhì)LTQ質(zhì)譜鑒定其組成成分。本文通過4次獨(dú)立重復(fù)實(shí)驗(yàn)共獲得53個相互作用數(shù)據(jù),生物信息學(xué)分析發(fā)現(xiàn),其中與HNF3β共定位的蛋白質(zhì)有9個,具有相互作用結(jié)構(gòu)域的的蛋白質(zhì)有5個,有10個分子同靶蛋白具有相同GO注釋,有9個分子與靶蛋白之間存在間接相互作用。基于這些分析結(jié)果,挑選其中4個相互作用分子,構(gòu)建真核表達(dá)載體,進(jìn)行報(bào)告基因?qū)嶒?yàn)和外源性免疫共沉淀實(shí)驗(yàn)的驗(yàn)證。我們驗(yàn)證了LMNA與HNF3β的相互作用,報(bào)道基因?qū)嶒?yàn)表明LMNA可以影響HNF3β的轉(zhuǎn)錄活性。免疫共聚焦實(shí)驗(yàn)發(fā)現(xiàn)LMNA與HNF3β共定位于細(xì)胞核,SiLMNA下調(diào)HepG2細(xì)胞內(nèi)LMNA的表達(dá),可以改變HNF3β下游靶基因的表達(dá)。 基于這些實(shí)驗(yàn)可推測HNF3β可和細(xì)胞內(nèi)多種蛋白質(zhì)發(fā)揮作用,協(xié)同參與轉(zhuǎn)錄調(diào)控, LMNA-HNF3β之間相互作用的實(shí)驗(yàn)研究表明它們之間的相互作用可能具有重要的生理意義,并可能在葡萄糖、脂肪代謝及相關(guān)疾病的發(fā)生、發(fā)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.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R341
本文編號：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.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R341
【共引文獻(xiàn)】
相關(guān)期刊論文 前2條
1 陸明;劉麗梅;;翼螺旋轉(zhuǎn)錄因子Foxa2與糖脂代謝及糖尿病關(guān)系的研究進(jìn)展[J];上海交通大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2011年04期
2 孫婷婷;宋麗娜;于淼;李長燕;楊曉明;汪思應(yīng);;免疫共沉淀聯(lián)合質(zhì)譜對肝細(xì)胞核因子3β蛋白復(fù)合體的分離鑒定[J];生物技術(shù)通訊;2011年05期
，本文編號：2336918
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