Characterising plant deubiquitinases with in vitro activity-based labelling and ubiquitin chain disassembly assays

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Abstract

Post-translational modification of proteins by ubiquitin is an essential cellular signaling mechanism in all eukaryotes. Ubiquitin is removed from target proteins by a wide range of deubiquitinase (DUB) enzymes with different activities and substrate specificities. Understanding how DUBs function in vitro is a vital first step to uncovering their cellular roles. Here we provide protocols for rapidly analyzing DUB activity in vitro by activity-based labelling with the suicide probe HA-ubiquitin vinyl sulfone (HA-UbVS) and by ubiquitin chain disassembly assays. We have previously used these methods to analyses the activity of the Arabidopsis thaliana DUB, UBP6, but these protocols are in principle applicable to any DUB of interest.
Original languageEnglish
Article numbere4015
Number of pages5
JournalBio-protocol
Volume11
Issue number9
DOIs
Publication statusPublished - 5 May 2021

Keywords

  • ubiquitin
  • deubiquitinase
  • ubiquitin vinyl sulfone
  • proteasome
  • cell signaling
  • proteostasis

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