Dataset Information

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

ABSTRACT: The methionine1 (M1)-specific deubiquitinase OTULIN acts as a negative regulator of NF-ΚB signaling and immune homeostasis. By replacing G76 in distal ubiquitin by dehydroalanine we designed the diubiquitin (diUb) probe UbG76Dha-Ub (OTULIN ABP) that couples to the catalytic site of OTULIN and thereby captures OTULIN in its active conformation. OTULIN ABP displays high selectivity for OTULIN and is not labeling to other M1-specific DUB including CYLD. In fact, labeling of USP5/IsopeptidaseT was the only detectable cross-reactivity. Further, ATP-dependent association of UbG76Dha-Ub probe with E1 activating enzymes induces assembly of polyOTULIN ABP chains, but USP5 labelling and E1 binding was abolished by deleting the C-terminal glycine in proximal ubiquitin (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 DUB for OTULIN that will facilitate studying the cellular function of this critical deubiquitinase.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Primary Cell

SUBMITTER: Philip Charles

LAB HEAD: Benedikt M Kessler

PROVIDER: PXD006868 | Pride | 2017-09-19

REPOSITORIES: Pride

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			Action	DRS
	HF247_MSQ1052_AureliaWeber_S1_1.raw	Raw
	HF247_MSQ1052_AureliaWeber_S1_2.raw	Raw
	HF247_MSQ1052_AureliaWeber_S1_3.raw	Raw
	HF247_MSQ1052_AureliaWeber_S1_4.raw	Raw
	HF247_MSQ1052_AureliaWeber_S2_1.raw	Raw

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Publications

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

Weber Aurelia A Elliott Paul R PR Pinto-Fernandez Adan A Bonham Sarah S Kessler Benedikt M BM Komander David D El Oualid Farid F Krappmann Daniel D

Cell chemical biology 20170914 10

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<sub>G76Dha</sub>-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 ...[more]

PMID: 28919039

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