鉴定cyclic di-GMP效应蛋白：高通量筛选策略与实验验证方法

环二鸟苷单磷酸(cyclic di-GMP或c-di-GMP)是细菌细胞中广泛存在的第二信使,调控细菌生物被膜发育、致病力、运动性、胞外多糖产生及细胞周期在内的诸多重要生理表型。c-di-GMP通过结合多种类型的效应子(包括核糖开关或效应蛋白)来发挥调控功能。由于c-di-GMP分子在构象上具有多变性,其结合的效应子同样具有多样性。新型效应蛋白的筛选、鉴定是当前细菌信号转导领域的研究热点和难点,也是解析c-di-GMP调控机制的首要环节。本文在阐述c-di-GMP结合不同类型的效应蛋白并调控细菌生物被膜发育的基础上,综述了目前筛选c-di-GMP效应蛋白的方法,包括遗传筛选、亲和色谱结合质谱鉴定、DRa CALA系统鉴定以及基于分子对接的预测等。同时,对验证c-di-GMP效应蛋白的技术,如等温微量热滴定、表面等离子共振、微量热泳动在内的多种验证方法进行了总结,对比了这些策略和方法在应用上的优、缺点,为在细菌及其真核宿主基因组水平鉴定c-di-GMP效应蛋白的研究提供参考。

Identification of cyclic di-GMP protein receptors: high-throughput screening strategies and experimental verification

cyclic di-GMP (c-di-GMP) is a universal second messenger in bacterial cells. It regulates various biological processes such as biofilm development, pathogenicity, motility, exopolysaccharide (EPS) production and cell cycle. The second messenger exerts its function by binding to effectors, such as riboswitches and proteins. However, due to the diverse conformations of c-di-GMP, its effectors are hardly to be predicted by homology search. Identification of c-di-GMP effectors is the initial step to investigate its regulatory function in bacterial signal transduction, however, it remains to be a technically difficult task. Here we reviewed the mechanism of biofilm development controlled by c-di-GMP through binding to various types of protein effectors, and summarized the screening strategies, including genetics analysis, protein pull-down combined with LC/MS/MS identification, DRaCALA systematic screening and molecular docking-based prediction. We also summarized experimental methods for verifying protein-c-di-GMP interaction, including isothermal titration calorimetry, surface plasmon resonance, microscale thermophoresis etc. In addition, we discussed the advantages and disadvantages of these strategies and methods. The present review aims to facilitate the future investigations that are focused on regulatory role of novel c-di-GMP effectors.