Structural and functional characterization of ubiquitin variant inhibitors of USP15

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. Taking advantage of the modular nature of ubiquitin, we designed a linear dimer (diUbV) consisting of a UbV that bound the DUSP domain followed by a UbV that bound the catalytic domain, which exhibited enhanced specificity and more potent inhibition of USP15 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 TGF- pathway. Structural analyses of three distinct UbVs bound to the catalytic domain revealed that the inhibitors locked the active site in a closed, inactive conformation. The structure of a UbV-DUSP domain complex revealed that the UbV formed an unusual strand-swapped dimer that bound two DUSP domains. These inhibitors will enable the study of USP15 function in vitro and in vivo to explore the role of the enzyme in oncology, neurology, immunology and inflammation.

多结构域去泛素化酶USP15可调控多种真核生物过程，并与诸多疾病的发生发展密切相关。我们开发了靶向USP15催化结构域，或是其三个衔接子结构域（含N端DUSP结构域）的泛素变体（ubiquitin variants，UbVs）。依托泛素的模块化特性，我们设计了一种线性二聚体泛素变体（diUbV）：由先结合DUSP结构域的UbV与结合催化结构域的UbV串联而成，相较于单一UbV，该二聚体对USP15催化活性的特异性更强、抑制效力更为显著。在细胞实验中，UbVs可抑制两种USP15底物SMURF2与TRIM25的去泛素化过程，而diUbV则能阻断USP15对转化生长因子-β（TGF-β）信号通路的调控作用。对三种结合催化结构域的不同UbV进行结构解析后发现，这些抑制剂可将USP15的活性位点锁定在闭合的非活化构象中。而UbV与DUSP结构域形成的复合物结构显示，UbV会形成一种罕见的链交换二聚体，可结合两个DUSP结构域。这些抑制剂将为体外及体内研究USP15的功能提供有力工具，助力探索该酶在肿瘤学、神经科学、免疫学及炎症领域的潜在作用。