Draganov Simeon D, Jones Hannah B L, Shah Qura, Kessler Benedikt M, Pinto-Fernández Adán
Chinese Academy of Medical Sciences Oxford Institute, Nuffield Department of Medicine, University of Oxford, Oxford, UK.
Target Discovery Institute, Centre for Medicines Discovery, Nuffield Department of Medicine, University of Oxford, Oxford, UK.
Methods Mol Biol. 2025;2921:1-29. doi: 10.1007/978-1-0716-4502-4_1.
A detailed methodology platform is described for activity-based protein profiling (ABPP) of cellular deISGylating enzymes using a specific activity-based interferon-stimulated gene 15 (ISG15) probe. Manual and semi-automated workflows for medium- to high-throughput applications are outlined in this chapter, with western blotting and proteomics-based techniques as the main readouts. This methodology informs us of endogenous deISGylating enzyme expression and activity in a cellular context, including USP18, the type I interferon (IFN-I)-inducible deISGylase, and several constitutively expressed deubiquitinases (DUBs), such as USP5, USP14, USP16, and USP36, that exert cross-reactivity to ISG15. ISG15-ABPP also enables the identification and characterization of potent and selective deISGylating enzyme modulators.
本文描述了一个详细的方法平台，用于使用基于活性的特异性干扰素刺激基因15（ISG15）探针，对细胞去ISGylation酶进行基于活性的蛋白质谱分析（ABPP）。本章概述了用于中高通量应用的手动和半自动工作流程，主要以蛋白质印迹法和基于蛋白质组学的技术作为检测手段。该方法让我们了解细胞内源性去ISGylation酶的表达和活性，包括I型干扰素（IFN-I）诱导的去ISGylase USP18，以及几种组成性表达的去泛素化酶（DUBs），如USP5、USP14、USP16和USP36，它们对ISG15具有交叉反应性。ISG15-ABPP还能够鉴定和表征强效且选择性的去ISGylation酶调节剂。
