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A Linear Diubiquitin-Based Probe for Efficient and Selective Detection of the Deubiquitinating Enzyme OTULIN.

Authors :
Weber A
Elliott PR
Pinto-Fernandez A
Bonham S
Kessler BM
Komander D
El Oualid F
Krappmann D
Source :
Cell chemical biology [Cell Chem Biol] 2017 Oct 19; Vol. 24 (10), pp. 1299-1313.e7. Date of Electronic Publication: 2017 Sep 14.
Publication Year :
2017

Abstract

The methionine 1 (M1)-specific deubiquitinase (DUB) OTULIN acts as a negative regulator of nuclear factor κB signaling and immune homeostasis. By replacing Gly76 in distal ubiquitin (Ub) by dehydroalanine we designed the diubiquitin (diUb) activity-based probe Ub <subscript>G76Dha</subscript> -Ub (OTULIN activity-based probe [ABP]) that couples to the catalytic site of OTULIN and thereby captures OTULIN in its active conformation. The OTULIN ABP displays high selectivity for OTULIN and does not label other M1-cleaving DUBs, including CYLD. The only detectable cross-reactivities were the labeling of USP5 (Isopeptidase T) and an ATP-dependent assembly of polyOTULIN ABP chains via Ub-activating E1 enzymes. Both cross-reactivities were abolished by the removal of the C-terminal Gly in the ABP's proximal Ub, yielding the specific OTULIN probe Ub <subscript>G76Dha</subscript> -Ub <subscript>ΔG76</subscript> (OTULIN ABPΔG76). Pull-downs demonstrate that substrate-bound OTULIN associates with the linear ubiquitin chain assembly complex (LUBAC). Thus, we present a highly selective ABP for OTULIN that will facilitate studying the cellular function of this essential DUB.<br /> (Copyright © 2017 MRC Laboratory of Molecular Biology. Published by Elsevier Ltd.. All rights reserved.)

Details

Language :
English
ISSN :
2451-9448
Volume :
24
Issue :
10
Database :
MEDLINE
Journal :
Cell chemical biology
Publication Type :
Academic Journal
Accession number :
28919039
Full Text :
https://doi.org/10.1016/j.chembiol.2017.08.006