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Related Experiment Video

Updated: Dec 7, 2025

Screening Traditional Chinese Medicine Compounds for Inhibiting UCHL3 Activity Based on Molecular Docking and Deubiquitinating Enzyme Probe Technology
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Proteomics-Based Identification of DUB Substrates Using Selective Inhibitors.

Jonathan W Bushman1, Katherine A Donovan1, Nathan J Schauer1

  • 1Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02215, USA; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.

Cell Chemical Biology
|October 2, 2020
PubMed
Summary

Researchers developed a new proteomic method using deubiquitinating enzyme (DUB) inhibitors to identify DUB substrates. This approach successfully mapped USP7 targets, revealing their roles in DNA repair and E3 ligase pathways, aiding therapeutic development.

Keywords:
DUBschemical probedeubiquitinating enzymesdrug discoveryproteomics

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Proteomics

Background:

  • Deubiquitinating enzymes (DUBs) remove ubiquitin, regulating protein levels and function, and are key therapeutic targets.
  • Identifying DUB substrates is crucial for understanding their roles, but faces technical challenges.
  • USP7 is a DUB of significant therapeutic interest, yet its substrates and functions are not fully characterized.

Purpose of the Study:

  • To establish a proteome-wide method for identifying DUB substrates using DUB inhibitors and mass spectrometry.
  • To comprehensively identify and annotate substrates of the deubiquitinating enzyme USP7.
  • To explore the functional roles of USP7 substrates in cellular processes.

Main Methods:

  • Treatment of cells with potent DUB inhibitors.
  • Application of mass spectrometry-based proteomics to identify DUB substrates.
  • Proteome-wide analysis to discover known and novel USP7 targets.

Main Results:

  • A novel proteomic strategy was developed to identify DUB substrates at a large scale.
  • Numerous known and novel substrates of USP7 were identified.
  • USP7 substrates were found to be significantly enriched in DNA repair enzymes and E3 ubiquitin ligases.

Conclusions:

  • The developed method provides a generalizable protocol for identifying DUB substrates across various DUBs.
  • This approach facilitates a deeper understanding of DUB functions and their involvement in cellular pathways.
  • The findings are critical for advancing the translational development of DUB-targeted therapies.