Photo-affinity pulling down of low-affinity binding proteins mediated by post-translational modifications
Photo-affinity pulling down of low-affinity binding proteins mediated by post-translational modifications
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翻译后修饰介导的低亲和力结合蛋白的光亲和力下拉
DOI：
10.1016/j.aca.2020.02.016
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发表时间：
2020
影响因子：
6.2
通讯作者：
Guo Zhigang
中科院分区：
文献类型：
--
作者：
Yang Yang;He Mengyuan;Wei Tianxiang;Sun Junhua;Wu Shaohua;Gao Tao;Guo Zhigang
由翻译后修饰（PTMs）介导的蛋白质-蛋白质弱相互作用（PPI）在生物系统中起着重要作用。然而，研究PTM介导的PPI的技术挑战阻碍了许多研究进展。在这项工作中，我们开发了一种光亲和下拉分析方法，以下拉低亲和力结合蛋白，从而筛选PTM介导的PPI。在该方法中，PTM介导的非共价相互作用可以通过光活化的键合转化为共价相互作用，从而使低亲和力结合相互作用定格。制备的光亲和磁珠（PAMB）确保了高特异性和分辨率，以捕获相互作用的蛋白质。此外，与传统的下拉法相比，在PAMB上引入PEG钝化层显著降低了非特异性相互作用。为了验证概念，通过使用这种新开发的检测方法，我们已经鉴定了一组可以与Flap Endonuclease 1（FEN 1）蛋白上的特定甲基化位点相互作用的蛋白质。与传统的生物素-亲和素下拉系统相比，更少的干扰蛋白（减少超过80%）和更多的蛋白质亚类被描绘。因此，这种新的下拉方法可能为低亲和力PPI的研究提供有用的工具，并有助于发现生物医学研究中根本需要的新PTM介导的相互作用的潜在靶点。
Weak and transient protein-protein interactions (PPIs) mediated by the post-translational modifications (PTMs) play key roles in biological systems. However, technical challenges to investigate the PTM-mediated PPIs have impeded many research advances. In this work, we develop a photo-affinity pull-down assay method to pull-down low-affinity binding proteins, thus for the screen of PTM-mediated PPIs. In this method, the PTM-mediated non-covalent interactions can be converted to the covalent interactions by the photo-activated linkage, so as to freeze frame the low-affinity binding interactions. The fabricated photo-affinity magnetic beads (PAMBs) ensure high specificity and resolution to capture the interacted proteins. Besides, the introduction of PEG passivation layer on PAMB has significantly reduced the non-specific interaction as compared to the traditional pull-down assay. For proof-of-concept, by using this newly developed assay method, we have identified a set of proteins that can interact with a specific methylation site on Flap Endonuclease 1 (FEN1) protein. Less interfering proteins (decreased over 80%) and more proteins sub-classes are profiled as compared to the traditional biotin-avidin pull-down system. Therefore, this new pull-down method may provide a useful tool for the study of low-affinity PPIs, and contribute to the discovery of potential targets for renewed PTM-mediated interactions that is fundamentally needed in biomedical research.