JoVE - Journal of Visualized Experiments
JoVE Visualize
Research Domains
View All

Contact Us

Activity-Based Protein Profiling (ABPP) of Cellular DeISGylating Enzymes and Inhibitor Screening

  • 1Chinese Academy of Medical Sciences Oxford Institute, Nuffield Department of Medicine, University of Oxford, Oxford, UK. simeon.draganov@ndm.ox.ac.uk.
  • 2Target Discovery Institute, Centre for Medicines Discovery, Nuffield Department of Medicine, University of Oxford, Oxford, UK. simeon.draganov@ndm.ox.ac.uk.
  • 3Target Discovery Institute, Centre for Medicines Discovery, Nuffield Department of Medicine, University of Oxford, Oxford, UK.
  • 4Life Sciences Solutions, Thermo Fisher Scientific, Paisley, UK.
  • 5Chinese Academy of Medical Sciences Oxford Institute, Nuffield Department of Medicine, University of Oxford, Oxford, UK.
  • 6Chinese Academy of Medical Sciences Oxford Institute, Nuffield Department of Medicine, University of Oxford, Oxford, UK. adan.pintofernandez@ndm.ox.ac.uk.
  • 7Target Discovery Institute, Centre for Medicines Discovery, Nuffield Department of Medicine, University of Oxford, Oxford, UK. adan.pintofernandez@ndm.ox.ac.uk.
+

|

June 14, 2025

Abstract

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.

Keywords:

Related Concept Videos