Development of Ubiquitin‐Based Probe for Metalloprotease Deubiquitinases

• Published in: Angewandte Chemie International Edition 2019-10, Vol.58 (41), p.14477-14482
• Main Authors: Hameed, Dharjath S., Sapmaz, Aysegul, Burggraaff, Lindsey, Amore, Alessia, Slingerland, Cornelis J., Westen, Gerard J. P., Ovaa, Huib
• Format: Article
• Language: English
• Subjects: biological activity; chelates; Chelation; Enzymes; Fluorescence; metalloenzymes; Metalloproteinase; Probes; Proteasome 26S; Proteins; Ubiquitin; Zinc
• ISSN: 1433-7851; 1521-3773
• DOI: 10.1002/anie.201906790

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. Trapped: The metalloprotease class of deubiquitinases (DUBs) is vital to many cellular processes, but not much is known about this class because of the lack of specific probes. These metalloenzymes have zinc in their active site that can be trapped with a chelation‐based activity probe. Described here is a chemically synthesized Ub‐based probe containing a zinc chelator that can bind to and pull‐down metalloprotease DUBs from mammalian cell lysates.