Yeast two-hybrid screening of proteins interacting with plasmin receptor subunit: C-terminal fragment of annexin A2
Yeast two-hybrid screening of proteins interacting with plasmin receptor subunit: C-terminal fragment of annexin A2
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DOI：
10.1038/aps.2011.121
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发表时间：
2011-11-01
影响因子：
8.2
通讯作者：
Simmet, Thomas
中科院分区：
文献类型：
--
作者：
Li, Qun;Laumonnier, Yves;Simmet, Thomas
目的：鉴定与膜联蛋白A2 (A2IC) c端片段相互作用的蛋白，该片段是由纤溶蛋白切割纤联蛋白受体（一种含有膜联蛋白A2的异源四聚体（AA2t））产生的。方法：从人单核细胞分离的cDNA中pcr扩增获得A2IC片段的编码基因，利用DNA连接试剂盒将其连接到pBTM116载体中。所得质粒（pBTM116-A2IC）用ABI PRISM 310基因分析仪测序。Western blot检测融合LexA-DNA结合域（BD）的A2IC诱饵蛋白的表达。利用酵母双杂交筛选技术鉴定与A2IC相互作用并编码于人类单核细胞cDNA文库中的蛋白。使用ABI PRISM BigDye终止周期测序ready反应试剂盒测定相关cdna的DNA序列。利用BLAST搜索分析（http://www.ncbi.nlm.nih.gov）在核苷酸序列数据库中搜索同源序列。通过小规模酵母转化和X-gal实验，确定了LexA-A2IC蛋白与组织蛋白酶S和SNX17的相互作用。结果：通过重组含有gal4活性结构域（GAL4-AD）的全长转录因子，采用酵母双杂交的方法，用人单核细胞cDNA文库筛选了经编码诱饵蛋白质粒转化的酵母。筛选1 × 10(7)个克隆后，鉴定出23个独立的β - gal阳性克隆。序列分析和数据库检索显示，其中15个阳性克隆匹配8种不同的蛋白（SNX17、ProCathepsin S、RPS2、ZBTB4、OGDH、CCDC32、PAPD4和已知与膜联蛋白A2相互作用的actin）。结论：A2IC与多种蛋白相互作用形成蛋白复合物，可能参与了纤溶酶诱导单核细胞活化的分子机制。酵母双杂交系统是研究蛋白质相互作用的有效方法。
Aim: To identify proteins that interact with the C-terminal fragment of annexin A2 (A2IC), generated by plasmin cleavage of the plasmin receptor, a heterotetramer (AA2t) containing annexin A2.Methods: The gene that encodes the A2IC fragment was obtained from PCR-amplified cDNA isolated from human monocytes, and was ligated into the pBTM116 vector using a DNA ligation kit. The resultant plasmid (pBTM116-A2IC) was sequenced with an ABI PRISM 310 Genetic Analyzer. The expression of an A2IC bait protein fused with a LexA-DNA binding domain (BD) was determined using Western blot analysis. The identification of proteins that interact with A2IC and are encoded in a human monocyte cDNA library was performed using yeast two-hybrid screening. The DNA sequences of the relevant cDNAs were determined using an ABI PRISM BigDye terminator cycle sequencing ready reaction kit. Nucleotide sequence databases were searched for homologous sequences using BLAST search analysis (http://www.ncbi.nlm.nih.gov). Confirmation of the interaction between the protein LexA-A2IC and each of cathepsin S and SNX17 was conducted using a small-scale yeast transformation and X-gal assay.Results: The yeast transformed with plasmids encoding the bait proteins were screened with a human monocyte cDNA library by reconstituting full-length transcription factors containing the GAL4-active domain (GAL4-AD) as the prey in a yeast two-hybrid approach. After screening 1x10(7) clones, 23 independent beta-Gal-positive clones were identified. Sequence analysis and a database search revealed that 15 of these positive clones matched eight different proteins (SNX17, ProCathepsin S, RPS2, ZBTB4, OGDH, CCDC32, PAPD4, and actin which was already known to interact with annexin A2).Conclusion: A2IC A2IC interacts with various proteins to form protein complexes, which may contribute to the molecular mechanism of monocyte activation induced by plasmin. The yeast two-hybrid system is an efficient approach for investigating protein interactions.