Department of Molecular Biology, Faculty of Science, Radboud Institute for Molecular Life Sciences, Radboud University Nijmegen, Nijmegen 6525, the Netherlands.
Department of Molecular Biology, Faculty of Science, Radboud Institute for Molecular Life Sciences, Radboud University Nijmegen, Nijmegen 6525, the Netherlands.
Mol Cell. 2017 Mar 2;65(5):941-955.e8. doi: 10.1016/j.molcel.2017.01.004. Epub 2017 Feb 9.
Intracellular signaling via the covalent attachment of different ubiquitin linkages to protein substrates is fundamental to many cellular processes. Although linkage-selective ubiquitin interactors have been studied on a case-by-case basis, proteome-wide analyses have not been conducted yet. Here, we present ubiquitin interactor affinity enrichment-mass spectrometry (UbIA-MS), a quantitative interaction proteomics method that makes use of chemically synthesized diubiquitin to enrich and identify ubiquitin linkage interactors from crude cell lysates. UbIA-MS reveals linkage-selective diubiquitin interactions in multiple cell types. For example, we identify TAB2 and TAB3 as novel K6 diubiquitin interactors and characterize UCHL3 as a K27-linkage selective interactor that regulates K27 polyubiquitin chain formation in cells. Additionally, we show a class of monoubiquitin and K6 diubiquitin interactors whose binding is induced by DNA damage. We expect that our proteome-wide diubiquitin interaction landscape and established workflows will have broad applications in the ongoing efforts to decipher the complex language of ubiquitin signaling.
细胞内信号通过不同的泛素连接键共价连接到蛋白质底物是许多细胞过程的基础。尽管已经对链接选择性泛素相互作用蛋白进行了逐个案例的研究，但尚未进行蛋白质组范围内的分析。在这里，我们提出了泛素相互作用体亲和富集 - 质谱（UbIA-MS），这是一种定量相互作用蛋白质组学方法，利用化学合成的二泛素从粗细胞裂解物中富集和鉴定泛素连接相互作用蛋白。UbIA-MS 揭示了多种细胞类型中链接选择性二泛素相互作用。例如，我们确定 TAB2 和 TAB3 为新型 K6 二泛素相互作用蛋白，并将 UCHL3 表征为 K27 连接选择性相互作用蛋白，该蛋白调节细胞中 K27 多泛素链的形成。此外，我们展示了一类单泛素和 K6 二泛素相互作用蛋白，其结合是由 DNA 损伤诱导的。我们期望我们的蛋白质组范围内的二泛素相互作用图谱和已建立的工作流程将在破译泛素信号的复杂语言的持续努力中具有广泛的应用。
