A linear diubiquitin-based probe for efficient and selective detection of the deubiquitinating enzyme OTULIN
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 prob...
| Main Authors: | , , , , , , , |
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| Format: | Journal article |
| Language: | English |
| Published: |
Cell Press
2017
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| _version_ | 1797084195295592448 |
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| author | Weber, A Elliott, P Pinto-Fernandez, A Bonham, S Kessler, B Komander, D El Oualid, F Krappmann, D |
| author_facet | Weber, A Elliott, P Pinto-Fernandez, A Bonham, S Kessler, B Komander, D El Oualid, F Krappmann, D |
| author_sort | Weber, A |
| collection | OXFORD |
| description | 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. |
| first_indexed | 2024-03-07T01:51:59Z |
| format | Journal article |
| id | oxford-uuid:9a6bd7bf-091a-42f0-86fa-2a1794f5959f |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T01:51:59Z |
| publishDate | 2017 |
| publisher | Cell Press |
| record_format | dspace |
| spelling | oxford-uuid:9a6bd7bf-091a-42f0-86fa-2a1794f5959f2022-03-27T00:21:12ZA linear diubiquitin-based probe for efficient and selective detection of the deubiquitinating enzyme OTULINJournal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:9a6bd7bf-091a-42f0-86fa-2a1794f5959fEnglishSymplectic Elements at OxfordCell Press2017Weber, AElliott, PPinto-Fernandez, ABonham, SKessler, BKomander, DEl Oualid, FKrappmann, DThe 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. |
| spellingShingle | Weber, A Elliott, P Pinto-Fernandez, A Bonham, S Kessler, B Komander, D El Oualid, F Krappmann, D A linear diubiquitin-based probe for efficient and selective detection of the deubiquitinating enzyme OTULIN |
| title | A linear diubiquitin-based probe for efficient and selective detection of the deubiquitinating enzyme OTULIN |
| title_full | A linear diubiquitin-based probe for efficient and selective detection of the deubiquitinating enzyme OTULIN |
| title_fullStr | A linear diubiquitin-based probe for efficient and selective detection of the deubiquitinating enzyme OTULIN |
| title_full_unstemmed | A linear diubiquitin-based probe for efficient and selective detection of the deubiquitinating enzyme OTULIN |
| title_short | A linear diubiquitin-based probe for efficient and selective detection of the deubiquitinating enzyme OTULIN |
| title_sort | linear diubiquitin based probe for efficient and selective detection of the deubiquitinating enzyme otulin |
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