Division of Biochemistry and Oncode Institute, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX, Amsterdam, the Netherlands.
Division of Cell Biology II, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX, Amsterdam, the Netherlands.
Nat Commun. 2019 Jan 16;10(1):231. doi: 10.1038/s41467-018-08231-5.
USP7 is a highly abundant deubiquitinating enzyme (DUB), involved in cellular processes including DNA damage response and apoptosis. USP7 has an unusual catalytic mechanism, where the low intrinsic activity of the catalytic domain (CD) increases when the C-terminal Ubl domains (Ubl45) fold onto the CD, allowing binding of the activating C-terminal tail near the catalytic site. Here we delineate how the target protein promotes the activation of USP7. Using NMR analysis and biochemistry we describe the order of activation steps, showing that ubiquitin binding is an instrumental step in USP7 activation. Using chemically synthesised p53-peptides we also demonstrate how the correct ubiquitinated substrate increases catalytic activity. We then used transient reaction kinetic modelling to define how the USP7 multistep mechanism is driven by target recognition. Our data show how this pleiotropic DUB can gain specificity for its cellular targets.
USP7 是一种高度丰富的去泛素化酶（DUB），参与包括 DNA 损伤反应和细胞凋亡在内的细胞过程。USP7 具有一种不寻常的催化机制，其催化结构域（CD）的固有低活性在 C 末端 Ubl 结构域（Ubl45）折叠到 CD 上时增加，从而允许激活的 C 末端尾巴靠近催化位点结合。在这里，我们描述了靶蛋白如何促进 USP7 的激活。我们使用 NMR 分析和生物化学描述了激活步骤的顺序，表明泛素结合是 USP7 激活的一个重要步骤。我们还使用化学合成的 p53 肽证明了正确泛素化的底物如何增加催化活性。然后，我们使用瞬时反应动力学模型来定义 USP7 多步骤机制如何被靶标识别所驱动。我们的数据表明，这种多效性的 DUB 如何获得其细胞靶标的特异性。
