Discovery of peptide ligands targeting a specific ubiquitin-like domain-binding site in the deubiquitinase USP11.
Discovery of peptide ligands targeting a specific ubiquitin-like domain-binding site in the deubiquitinase USP11.
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DOI：
10.1074/jbc.ra118.004469
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发表时间：
2019-01-11
期刊：
影响因子：
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通讯作者：
Dreveny I
中科院分区：
文献类型：
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作者：
Spiliotopoulos A;Blokpoel Ferreras L;Densham RM;Caulton SG;Maddison BC;Morris JR;Dixon JE;Gough KC;Dreveny I
泛素特异性蛋白酶 (USP) 可逆转泛素化并调节几乎所有细胞过程。已知 USP4 和 USP15 中定义的非催化结构域与 E3 连接酶和底物招募因子相互作用。旁系同源物 USP11 中的这些结构域尚未有此类相互作用的报道，USP11 是通过同源重组修复 DNA 双链断裂的关键调节因子。我们假设与其蛋白酶结构域相邻的 USP11 结构域具有独特的肽结合位点。在这里，我们使用下一代噬菌体展示 (NGPD) 策略，将噬菌体展示文库筛选与下一代测序相结合，发现了独特的 USP11 相互作用肽基序。等温滴定量热法表明，在体外，最高亲和力的肽（KD 约为 10 μm）对 USP11 表现出比 USP4 和 USP15 更独特的选择性。此外，USP11-肽复合物的晶体结构揭示了USP11非催化泛素样(UBL)区域中以前未知的结合位点。该位点与螺旋基序相互作用，并且在 USP4 和 USP15 中不存在。使用 USP11-WT 与结合袋缺陷双突变体进行的报告分析表明，该结合位点调节 USP11 在同源重组介导的 DNA 修复中的功能。与细胞递送序列融合的最高亲和力 USP11 肽结合剂诱导显着的核定位和 S 期细胞周期停滞，影响不同哺乳动物细胞系的活力。此处鉴定的 USP11 肽配体和 USP11 中旁系同源物特异性功能位点为开发新的生化工具和治疗剂提供了框架。我们建议基于 NGPD 的识别相互作用肽的策略也可以应用于其他细胞靶标。
Ubiquitin-specific proteases (USPs) reverse ubiquitination and regulate virtually all cellular processes. Defined noncatalytic domains in USP4 and USP15 are known to interact with E3 ligases and substrate recruitment factors. No such interactions have been reported for these domains in the paralog USP11, a key regulator of DNA double-strand break repair by homologous recombination. We hypothesized that USP11 domains adjacent to its protease domain harbor unique peptide-binding sites. Here, using a next-generation phage display (NGPD) strategy, combining phage display library screening with next-generation sequencing, we discovered unique USP11-interacting peptide motifs. Isothermal titration calorimetry disclosed that the highest affinity peptides (KD of ∼10 μm) exhibit exclusive selectivity for USP11 over USP4 and USP15 in vitro. Furthermore, a crystal structure of a USP11–peptide complex revealed a previously unknown binding site in USP11's noncatalytic ubiquitin-like (UBL) region. This site interacted with a helical motif and is absent in USP4 and USP15. Reporter assays using USP11-WT versus a binding pocket–deficient double mutant disclosed that this binding site modulates USP11's function in homologous recombination–mediated DNA repair. The highest affinity USP11 peptide binder fused to a cellular delivery sequence induced significant nuclear localization and cell cycle arrest in S phase, affecting the viability of different mammalian cell lines. The USP11 peptide ligands and the paralog-specific functional site in USP11 identified here provide a framework for the development of new biochemical tools and therapeutic agents. We propose that an NGPD-based strategy for identifying interacting peptides may be applied also to other cellular targets.