中文摘要：由cAMP1直接激活的交换蛋白（EPAC1）是cAMP的细胞内传感器，参与健康和疾病的多种细胞和生理过程。然而，目前还缺乏研究其与细胞内cAMP纳米结构域关系的相关试剂。本研究使用非抗体Affimer蛋白支架来开发EPAC1的异构体选择性蛋白结合剂。对纯化的、生物素化的人重组EPAC1 δDEP蛋白（氨基酸149-811）进行噬菌体展示筛选，鉴定出5种潜在的EPAC1选择性Affimer结合剂。然后用斑点杂交和间接ELISA分析确定Affimer 780A为顶部EPAC1结合剂。此外，研究显示780A能共沉淀转染细胞中的EPAC1，并分别与野生型EPAC1和错误靶向的EPAC1突变体（K212R）共定位，分别集中于于细胞的核周区和胞浆区。可见，作为一种新型的EPAC1选择性结合物，780A可在未来的研究中用于进一步了解cAMP-EPAC1信号系统的区域化。
外文摘要：An exchange protein directly activated by cAMP 1 (EPAC1) is an intracellular sensor for cAMP that is involved in a wide variety of cellular and physiological processes in health and disease. However, reagents are lacking to study its association with intracellular cAMP nanodomains. Here, we use non-antibody Affimer protein scaffolds to develop isoform-selective protein binders of EPAC1. Phage-display screens were carried out against purified, biotinylated human recombinant EPAC1 Delta DEP protein (amino acids 149-811), which identified five potential EPAC1-selective Affimer binders. Dot blots and indirect ELISA assays were next used to identify Affimer 780A as the top EPAC1 binder. Mutagenesis studies further revealed a potential interaction site for 780A within the EPAC1 cyclic nucleotide binding domain (CNBD). In addition, 780A was shown to co-precipitate EPAC1 from transfected cells and co-localize with both wild-type EPAC1 and a mis-targeting mutant of EPAC1(K212R), predominantly in perinuclear and cytosolic regions of cells, respectively. As a novel EPAC1-selective binder, 780A therefore has the potential to be used in future studies to further understand compartmentalization of the cAMP-EPAC1 signaling system.
外文关键词：EPAC1；cyclic AMP；affimer；microscopy；protein interactions；phage display
作者：Buist, HK；Luchowska-Stanska, U；van Basten, B；Valli, J；Smith, BO；Baillie, GS；Rickman, C；Ricketts, B；Davidson, A；Hannam, R；Sunderland, J；Yarwood, SJ
作者单位：Heriot Watt Univ；Univ Glasgow；Avacta Life Sci
期刊名称：CELLS
期刊影响因子：4.366
出版年份：2021
出版刊次：9
点击下载：鉴定并表征用于检测cAMP传感器EPAC1的Affimer亲和试剂