The Donnelly Centre, University of Toronto, Toronto, ON M5S 3E1, Canada.
Cancer Science Institute of Singapore, National University of Singapore, Singapore, Singapore.
Structure. 2019 Apr 2;27(4):590-605.e5. doi: 10.1016/j.str.2019.01.002. Epub 2019 Jan 31.
The multi-domain deubiquitinase USP15 regulates diverse eukaryotic processes and has been implicated in numerous diseases. We developed ubiquitin variants (UbVs) that targeted either the catalytic domain or each of three adaptor domains in USP15, including the N-terminal DUSP domain. We also designed a linear dimer (diUbV), which targeted the DUSP and catalytic domains, and exhibited enhanced specificity and more potent inhibition of catalytic activity than either UbV alone. In cells, the UbVs inhibited the deubiquitination of two USP15 substrates, SMURF2 and TRIM25, and the diUbV inhibited the effects of USP15 on the transforming growth factor β pathway. Structural analyses revealed that three distinct UbVs bound to the catalytic domain and locked the active site in a closed, inactive conformation, and one UbV formed an unusual strand-swapped dimer and bound two DUSP domains simultaneously. These inhibitors will enable the study of USP15 function in oncology, neurology, immunology, and inflammation.
多结构域去泛素化酶 USP15 调节多种真核生物过程，并与许多疾病相关。我们开发了靶向 USP15 的催化结构域或三个衔接结构域（包括 N 端的 DUSP 结构域）之一的泛素变体 (UbV)。我们还设计了一种线性二聚体 (diUbV)，其靶向 DUSP 和催化结构域，与单独的 UbV 相比，其特异性更强，对催化活性的抑制作用更强。在细胞中，UbV 抑制了 USP15 的两个底物 SMURF2 和 TRIM25 的去泛素化作用，而 diUbV 抑制了 USP15 对转化生长因子β途径的影响。结构分析表明，三种不同的 UbV 结合到催化结构域并将活性位点锁定在封闭的、无活性的构象中，而一种 UbV 形成了一种不寻常的链交换二聚体并同时结合两个 DUSP 结构域。这些抑制剂将使我们能够研究 USP15 在肿瘤学、神经学、免疫学和炎症中的功能。
