Development of Ubiquitin-Based Probe for Metalloprotease Deubiquitinases

Dharjath S. Hameed, Aysegul Sapmaz, Lindsey Burggraaff, Alessia Amore, Cornelis J. Slingerland, Gerard J.P. van Westen, Huib Ovaa*

*Corresponding author for this work

Research output: Contribution to JournalArticleAcademicpeer-review

Abstract

Deubiquitinases (DUBs) are a family of enzymes that regulate the ubiquitin signaling cascade by removing ubiquitin from specific proteins in response to distinct signals. DUBs that belong to the metalloprotease family (metalloDUBs) contain Zn2+ in their active sites and are an integral part of distinct cellular protein complexes. Little is known about these enzymes because of the lack of specific probes. Described here is a Ub-based probe that contains a ubiquitin moiety modified at its C-terminus with a Zn2+ chelating group based on 8-mercaptoquinoline, and a modification at the N-terminus with either a fluorescent tag or a pull-down tag. The probe is validated using Rpn11, a metalloDUB found in the 26S proteasome complex. This probe binds to metalloDUBs and efficiently pulled down overexpressed metalloDUBs from a HeLa cell lysate. Such probes may be used to study the mechanism of metalloDUBs in detail and allow better understanding of their biochemical processes.

Original languageEnglish
Pages (from-to)14477-14482
Number of pages6
JournalAngewandte Chemie. International Edition
Volume58
Issue number41
DOIs
Publication statusPublished - 7 Oct 2019
Externally publishedYes

Keywords

  • biological activity
  • chelates
  • metalloenzymes
  • ubiquitin
  • zinc

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