人ATP结合盒超家族蛋白相互作用蛋白的规模化发掘

发布时间：2018-06-10 05:06

本文选题：酵母双杂交 + ATP结合盒超家族转运蛋白　；参考：《第三军医大学》2006年硕士论文

【摘要】： ATP结合盒超家族转运蛋白[The ATP-binding cassette (ABC) transporter superfamily]属于一类分布广泛、数目众多的跨膜转运体系。它们可以转运代谢产物、离子、糖、氨基酸、脂类、固醇以及药物等多种底物通过细胞内、外膜。一些特定的ABC转运蛋白在多药耐性的肿瘤细胞系或癌症过表达,与临床耐药密切相关。这些基因的突变可能直接和间接导致多种遗传疾病。而目前关于ATP结合盒超家族转运蛋白具体转运机制以及功能还未完全了解。关于其与细胞内蛋白的相互作用研究甚少。规模化进行蛋白质相互作用研究可能发现其更精确的转运调节机制并揭示新的功能。蛋白质-蛋白质相互作用是一切生命活动的基础,揭示相互作用规律是认识生命奥秘的必经之路。酵母双杂交技术是目前应用最成功的大规模研究蛋白质相互作用的技术平台,规模化酵母双杂交技术不是常规实验室酵母双杂交技术在诱饵数量上的简单增加,需要解决大量的技术问题。本文以ATP结合盒超家族转运蛋白为研究对象,摸索规模化酵母双杂交研究蛋白质相互作用的技术条件,优化各关键技术环节,完善可用于规模化蛋白相互作用研究的酵母双杂交技术平台;发现20对高可信的蛋白相互作用。结果对采用酵母双杂交技术规模化研究蛋白质相互作用提供可借鉴的经验,发现的相互作用有助于揭示ABC蛋白的转运机制,并为其新功能的认识提供重要的功能线索。本文采用酵母双杂交技术,选取具有重要功能,代表6类亚家族的14种ATP结合盒超家族转运蛋白ABCA1、ABCA5、ABCA6、ABCB1、ABCB11、ABCB4、ABCC1、ABCC13、ABCC2、ABCC3、ABCC5、ABCC6、ABCF1、ABCG8的胞质区(C端或者N端)为诱饵,筛选成人肝cDNA文库,得到67种不同的相互作用。在对相互作用数据的初步分析中,除去不符合读框和常见假阳性,经过回转验证,得到20对可信度较高的相互作用。这些相互作用提示ATP结合盒超家族转运蛋白可能有许多新的功能,同时也揭示可能存在共同的转运机制。如发现ABCC2与NF-κB1的相互作用,提示ABBC2可能作为一个受体,参与NF-κB信号通路的激活和调节。用GO(gene ontology)进行功能注释,发现相互作用蛋白中有参与物质转运过程的,
[Abstract]:ATP-binding cassette transporter (ATP-binding cassette) transporter superfamily is a widely distributed and numerous transmembrane transporter system. They transport metabolites, ions, sugars, amino acids, lipids, steroids and drugs through the intracellular and outer membranes. Some specific ABC transporters are overexpressed in multidrug resistant tumor cell lines or cancer, which is closely related to clinical drug resistance. Mutations in these genes can lead directly and indirectly to multiple genetic diseases. However, the mechanism and function of ATP binding cassette superfamily transporters have not been fully understood. Little research has been done on the interaction between intracellular proteins and them. A large-scale study of protein interactions may reveal more precise mechanisms of transport regulation and reveal new functions. Protein-protein interactions are the basis of all life activities. Revealing the law of interaction is the only way to understand the mystery of life. Yeast two-hybrid technology is the most successful platform for studying protein interaction on a large scale at present. Large-scale yeast two-hybrid technology is not a simple increase in the number of decoys of yeast two-hybrid technology in conventional laboratory. A large number of technical problems need to be solved. In this paper, ATP-binding cassette superfamily transporters were used to explore the technical conditions for the study of protein interaction in large-scale yeast two-hybrid, and to optimize the key technical links. To improve the yeast two-hybrid technology platform for the study of large-scale protein interaction, 20 pairs of highly credible protein interactions were found. Results the experience of using yeast two-hybrid technique to study protein interaction on a large scale was provided, and the discovered interaction was helpful to reveal the transport mechanism of ABC protein. In this paper, yeast two-hybrid technique was used to select 14 ATP-binding box superfamily transporter proteins ABCA1, ABCA6, ABCB1, ABCB11, ABCC4, ABCC13, ABCC3, ABCC5, ABCA1, ABCA5, AB@@ The adult liver cDNA library was screened and 67 different interactions were obtained. In the preliminary analysis of the interaction data, 20 pairs of interactions with high reliability were obtained by gyrological verification, except for non-conforming reading frames and common false positives. These interactions suggest that ATP-binding cassette superfamily transporters may have many new functions and that there may be a common transport mechanism. If the interaction between ABCC2 and NF- 魏 B1 is found, ABBC2 may be a receptor involved in the activation and regulation of NF- 魏 B signaling pathway.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R341

【共引文献】