Jump to main content
Jump to site search
Access to RSC content Close the message box

Continue to access RSC content when you are not at your institution. Follow our step-by-step guide.


Issue 40, 2018
Previous Article Next Article

Activity-based ubiquitin-protein probes reveal target protein specificity of deubiquitinating enzymes

Author affiliations

Abstract

Ubiquitination is an essential eukaryotic post-translational modification that regulates various cellular processes. The removal of ubiquitin from its target protein is catalyzed by deubiquitinating enzymes (DUBs). Although it was proposed that many DUBs specifically interact and recognize ubiquitinated proteins as substrates, more direct evidence is needed to support this notion. Here we report protein-targeting activity-based DUB probes that allowed the identification of DUBs recognizing monoubiquitinated proliferating cell nuclear antigen (PCNA) in Saccharomyces cerevisiae. This new class of DUB probes contain a Michael acceptor as a warhead between ubiquitin and the target protein PCNA through a linkage that mimics the native isopeptide bond. We selected two known and biologically relevant ubiquitination sites on PCNA to generate the DUB probes. This allowed us to interrogate the site-specific deubiquitination of a target protein by DUBs. DUBs were profiled in yeast cell lysates using the two Ub-PCNA DUB probes in conjunction with two control probes that contain a noncleavable linkage but no warhead. We identified yeast DUBs through pulldown coupled with quantitative mass spectrometry analysis of the pulled down proteins. Our results showed that specific yeast DUBs recognize monoubiquitinated PCNA and corroborated previous genetic study. We also identified DUBs as potential new deubiquitinase of PCNA. Remarkably, identified DUBs clearly distinguish the different modification sites on PCNA, thus supporting a high level of DUB specificity beyond the target protein identity.

Graphical abstract: Activity-based ubiquitin-protein probes reveal target protein specificity of deubiquitinating enzymes

Back to tab navigation

Supplementary files

Article information


Submitted
06 Apr 2018
Accepted
08 Aug 2018
First published
30 Aug 2018

This article is Open Access
All publication charges for this article have been paid for by the Royal Society of Chemistry

Chem. Sci., 2018,9, 7859-7865
Article type
Edge Article

Activity-based ubiquitin-protein probes reveal target protein specificity of deubiquitinating enzymes

P. Gong, G. A. Davidson, W. Gui, K. Yang, W. P. Bozza and Z. Zhuang, Chem. Sci., 2018, 9, 7859
DOI: 10.1039/C8SC01573B

This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Material from this article can be used in other publications provided that the correct acknowledgement is given with the reproduced material and it is not used for commercial purposes.

Reproduced material should be attributed as follows:

  • For reproduction of material from NJC:
    [Original citation] - Published by The Royal Society of Chemistry (RSC) on behalf of the Centre National de la Recherche Scientifique (CNRS) and the RSC.
  • For reproduction of material from PCCP:
    [Original citation] - Published by the PCCP Owner Societies.
  • For reproduction of material from PPS:
    [Original citation] - Published by The Royal Society of Chemistry (RSC) on behalf of the European Society for Photobiology, the European Photochemistry Association, and RSC.
  • For reproduction of material from all other RSC journals:
    [Original citation] - Published by The Royal Society of Chemistry.

Information about reproducing material from RSC articles with different licences is available on our Permission Requests page.


Social activity

Search articles by author

Spotlight

Advertisements