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Development of Ubiquitin‐Based Probe for Metalloprotease Deubiquitinases
Author(s) -
Hameed Dharjath S.,
Sapmaz Aysegul,
Burggraaff Lindsey,
Amore Alessia,
Slingerland Cornelis J.,
Westen Gerard J. P.,
Ovaa Huib
Publication year - 2019
Publication title -
angewandte chemie
Language(s) - English
Resource type - Journals
eISSN - 1521-3757
pISSN - 0044-8249
DOI - 10.1002/ange.201906790
Subject(s) - ubiquitin , deubiquitinating enzyme , chemistry , proteasome , biochemistry , metalloproteinase , hela , moiety , enzyme , microbiology and biotechnology , ubiquitin ligase , biology , cell , stereochemistry , gene
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 Zn 2+ 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 Zn 2+ 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.