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

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

  • Cell Chem Biol. 2017 Oct 19;24(10):1299-1313.e7. doi: 10.1016/j.chembiol.2017.08.006.
Aurelia Weber 1,  Paul R Elliott 2,  Adan Pinto-Fernandez 3,  Sarah Bonham 3,  Benedikt M Kessler 3,  David Komander 2,  Farid El Oualid 4,  Daniel Krappmann 5
Affiliations

Affiliations

  • 1 Research Unit Cellular Signal Integration, Institute of Molecular Toxicology and Pharmacology, Helmholtz Zentrum München - German Research Center for Environmental Health, Ingolstaedter Landstrasse 1, 85764 Neuherberg, Germany.
  • 2 Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.
  • 3 Target Discovery Institute, Nuffield Department of Medicine, University of Oxford, Roosevelt Drive, Oxford OX3 7FZ, UK.
  • 4 UbiQ Bio BV, Science Park 408, 1098 XH Amsterdam, the Netherlands. Electronic address: farideloualid@ubiqbio.com.
  • 5 Research Unit Cellular Signal Integration, Institute of Molecular Toxicology and Pharmacology, Helmholtz Zentrum München - German Research Center for Environmental Health, Ingolstaedter Landstrasse 1, 85764 Neuherberg, Germany. Electronic address: daniel.krappmann@helmholtz-muenchen.de.
PMID: 28919039 DOI: 10.1016/j.chembiol.2017.08.006
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 UbG76Dha-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 UbG76Dha-UbΔG76 (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.

Keywords

DUB; HOIP; M1-linked; OTULIN; activity-based probe; ubiquitin chains; ubiquitin-activating E1.

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