Development of a Ubiquitin based probe for metalloprotease deubiquitinases.

Abstract

The deubiquitinases (DUBs) are a family of enzymes that regulate the ubiquitin signalling cascade by removing ubiquitin from specific proteins in response to distinct signals. The DUBs that belong to the metalloprotease family (metalloDUBs) contain Zn2+ in its active site and are an integral part of distinct cellular protein complexes. Little is known about these enzymes due to the lack of specific probes because of the absence of a covalent enzyme-substrate intermediate complex during the deubiquitination process. Here we describe a Ub-based probe that contains a ubiquitin moiety modified at its C-terminus with a Zn2+ chelating group based on 8-mercaptoquinoline and that is modified at the N-terminus with a fluorescent tag or a pull-down tag. The probe is validated using Rpn11, a metalloDUB found in the 26S proteasome complex. This probe is able to bind to metalloDUBs and efficiently pulled down overexpressed metalloDUBs from HeLa cell lysate. Such probes may be used to study the mechanism of metalloDUBs in detail. This will allow us to better understand the biochemical processes of protein complexes that contain metalloDUBs.