Konstantinou Aimiliani, Varga Julia K, Córdova-Pérez Alicia, Simonetti Leandro, Gomez-Lucas Lidia, Schueler-Furman Ora, Davey Norman E, Kulathu Yogesh, Ivarsson Ylva
Department of Chemistry-BMC, Uppsala University, Box 576, Uppsala, 751 23, Sweden.
Department of Microbiology and Molecular Genetics, Institute for Biomedical Research IMRIC, Faculty of Medicine, Hebrew University of Jerusalem, Jerusalem, Israel.
Biol Direct. 2025 May 6;20(1):59. doi: 10.1186/s13062-025-00638-7.
Ubiquitin-specific protease 8 (USP8) is a deubiquitinating enzyme with essential functions in protein trafficking and stability. It is a multidomain protein, with an N-terminal MIT (microtubule interacting and trafficking) domain, followed by a non-catalytic rhodanese (Rhod) domain, a long intrinsically disordered region, and a C-terminal catalytic domain. The N-terminal MIT domain of USP8 is known to mediate protein-protein interactions through binding to short linear motifs. The non-catalytic Rhod domain is also involved in protein-protein interactions, however detailed insights into these interactions remain limited. In this study we explore the short linear motif-based interactions of the MIT and Rhod domains of USP8 using a combination of proteomic peptide-phage display, peptide arrays and deep mutational scanning. We show that the MIT domain can bind ligands with a general [DE][LIF]x{2,3}R[FYIL]xxL[LV] consensus motif. We uncover that the rhodanese domain of USP8 is a peptide-binding domain, and define two distinct binding motifs (Rx[LI]xGxxxPxxL and G[LV][DE][IM]WExKxxxLxE) for this domain by deep mutational scanning of two different peptide ligands. Using the motif information, we predict binding sites within known USP8 interactors and substrates and validate interactions through peptide array analysis. Our findings demonstrate that both the USP8 MIT and rhodanese domains are peptide-binding domains that can be bound by degenerate and distinct binding motifs. The detailed information on the peptide binding preference of the two N-terminal domains of USP8 provide novel insights into the molecular recognition events that underlie the function of this essential deubiquitinating enzyme.
泛素特异性蛋白酶8（USP8）是一种去泛素化酶，在蛋白质运输和稳定性方面具有重要功能。它是一种多结构域蛋白，具有一个N端的微管相互作用和运输（MIT）结构域，接着是一个非催化性的硫氧还蛋白（Rhod）结构域、一个长的内在无序区域以及一个C端催化结构域。已知USP8的N端MIT结构域通过与短线性基序结合来介导蛋白质-蛋白质相互作用。非催化性的Rhod结构域也参与蛋白质-蛋白质相互作用，然而对这些相互作用的详细了解仍然有限。在本研究中，我们结合蛋白质组学肽-噬菌体展示、肽阵列和深度突变扫描，探索USP8的MIT和Rhod结构域基于短线性基序的相互作用。我们表明，MIT结构域能够结合具有通用[DE][LIF]x{2,3}R[FYIL]xxL[LV]共有基序的配体。我们发现USP8的硫氧还蛋白结构域是一个肽结合结构域，并通过对两种不同肽配体的深度突变扫描为该结构域定义了两个不同的结合基序（Rx[LI]xGxxxPxxL和G[LV][DE][IM]WExKxxxLxE）。利用这些基序信息，我们预测了已知USP8相互作用蛋白和底物中的结合位点，并通过肽阵列分析验证了相互作用。我们的研究结果表明，USP8的MIT和硫氧还蛋白结构域都是肽结合结构域，可被简并和不同的结合基序所结合。关于USP8两个N端结构域肽结合偏好的详细信息，为这一重要去泛素化酶功能背后的分子识别事件提供了新的见解。
