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.
Journal article
2025-01-01T00:00:00+00:00
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Chinese Academy of Medical Sciences Oxford Institute, Nuffield Department of Medicine, University of Oxford, Oxford, UK. simeon.draganov@ndm.ox.ac.uk.
Humans, Ubiquitins, Cytokines, Enzyme Inhibitors, Blotting, Western, Proteomics, High-Throughput Screening Assays, Deubiquitinating Enzymes