Development of Ubiquitin‐Based Probe for Metalloprotease Deubiquitinases. (28th August 2019)

• Record Type: Journal Article
• Authors: Hameed, Dharjath S.; Sapmaz, Aysegul; Burggraaff, Lindsey; Amore, Alessia; Slingerland, Cornelis J.; van Westen, Gerard J. P.; Ovaa, Huib
• Is Part Of: Angewandte Chemie. Volume 131:Number 41(2019)
• Page Start: 14619
• Page End: 14624
• Publication Date: 2019-08-28
• Subjects: Biologische Aktivität -- Chelate -- Metalloenzyme -- Ubiquitin -- Zink; Chemistry -- Periodicals; 540
• DOI: 10.1002/ange.201906790
• Languages: English
• ISSNs: 0044-8249

Abstract

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 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. Abstract : Eingefangen : Die Metalloproteaseklasse der Deubiquitinasen (DUBs) ist essenziell für viele Zellprozesse, allerdings ist nicht viel über sie bekannt. Diese Metalloenzyme enthalten Zink in ihrem aktiven Zentrum, das mit einer chelatisierungsbasierten Aktivitätssonde eingefangen werden kann. Hier wird eine Ub‐basierte Sonde mit einem Zink‐Chelator vorgestellt, die an Metalloprotease‐DUBs aus Zell‐Lysaten von Säugetieren binden kann.