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Weber, A. ; Elliott, P.R.* ; Pinto-Fernandez, A.* ; Bonham, S.* ; Kessler, B.M.* ; Komander, D.* ; El Oualid, F.* ; Krappmann, D.

A linear diubiquitin-based probe for efficient and selective detection of the deubiquitinating enzyme OTULIN.

Cell Chem. Bio. 24, 1299-1313.e7 (2017)

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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.

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Publication type Article: Journal article

Document type Scientific Article

Corresponding Author

Keywords Dub ; Hoip ; M1-linked ; Otulin ; Activity-based Probe ; Ubiquitin Chains ; Ubiquitin-activating E1; Nf-kappa-b; Ubiquitin-based Probes; Spata2 Links Cyld; Cell-death; Met1-linked Ubiquitination; Polyubiquitin Chains; Signaling Complexes; Molecular-basis; Specificity; Binding

ISSN (print) / ISBN 2451-9448

e-ISSN 2451-9456

Journal Cell Chemical Biology

Quellenangaben Volume: 24, Issue: 10, Pages: 1299-1313.e7

Publisher Cell Press

Publishing Place Cambridge, Massachusetts

Non-patent literature Publications

Reviewing status Peer reviewed

Institute(s) Research Unit Signaling and Translation (SAT)