2011 Collaborative Innovation Center of Tianjin for Medical Epigenetics, Tianjin Key Laboratory of Medical Epigenetics, Key Laboratory of Immune Microenvironment and Disease (Ministry of Education), Tianjin Key Laboratory of Cellular Homeostasis and Diseases, School of Basic Medical Sciences, Tianjin Medical University, Tianjin 300070, China.
School of Biomedical Engineering, Tianjin Medical University, Tianjin 300070, China.
Anal Chem. 2020 May 5;92(9):6218-6223. doi: 10.1021/acs.analchem.9b05394. Epub 2020 Apr 14.
Histone post-translational modifications (HPTMs) serve as signal platforms for recruitment of binding proteins (readers) to regulate gene expression. Accumulated evidence suggests that the intensive distribution of HPTMs may result in crosstalk, which increases or inhibits the recruitment of reader proteins, further altering the functional outcome of HPTMs. Therefore, the comprehensive identification of multiple interactions between combinatorial HPTMs and reading domains is essential to understand the chromatin-templated processes. However, it is still a big challenge to profile these complicated interactions due to various limitations including rather weak, transient and multiple interactions between HPTMs and readers, the high dynamic property of HPTMs as well as the low abundance of reader proteins. Here we developed an integrated approach to profile the complicated interactions between combinatorial HPTMs and dual domains. Based on a combinatorial HPTM peptide library (trimethylation of histone H3 lysine 4 and its neighboring PTMs) and five affinity tag proteins containing tandem-domain probes, histone interactions can be profiled by pull-down assay combined with mass spectrometry analysis. The interactions were further verified by isothermal titration calorimetry and proximity ligation assay, as well as molecular docking. By use of combinatorial HPTMs, we demonstrated that this integrated approach can be successfully utilized for the characterization of multiple interactions between reading domains and combinatorial HPTMs including novel HPTMs with low stoichiometry. Thus, a novel chemical proteomics tool for profiling of multiple PTM-mediated protein-protein interactions was successfully developed and can be adapted for broad biomedical applications.
组蛋白翻译后修饰（HPTMs）作为信号平台，招募结合蛋白（阅读器）来调节基因表达。大量证据表明，HPTMs 的密集分布可能导致串扰，从而增加或抑制阅读器蛋白的募集，进一步改变 HPTMs 的功能结果。因此，全面识别组合 HPTMs 与阅读结构域之间的多种相互作用对于理解染色质模板化过程至关重要。然而，由于各种限制因素，包括 HPTMs 与阅读器之间的相互作用较弱、短暂且多样，HPTMs 的高动态特性以及阅读器蛋白的低丰度，全面分析这些复杂的相互作用仍然是一个巨大的挑战。在这里，我们开发了一种综合方法来分析组合 HPTMs 与双结构域之间的复杂相互作用。基于组合 HPTM 肽文库（组蛋白 H3 赖氨酸 4 的三甲基化及其相邻的 PTMs）和包含串联结构域探针的五种亲和标签蛋白，通过下拉测定结合质谱分析可以对组蛋白相互作用进行分析。通过等温滴定量热法和邻近连接测定以及分子对接进一步验证了相互作用。通过使用组合 HPTMs，我们证明了这种综合方法可以成功用于表征阅读结构域与组合 HPTMs 之间的多种相互作用，包括低计量的新型 HPTMs。因此，成功开发了一种用于分析多种 PTM 介导的蛋白质-蛋白质相互作用的新型化学蛋白质组学工具，并且可以适应广泛的生物医学应用。
