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A cascading activity-based probe sequentially targets E1-E2-E3 ubiquitin enzymes.

Authors :
Mulder MP
Witting K
Berlin I
Pruneda JN
Wu KP
Chang JG
Merkx R
Bialas J
Groettrup M
Vertegaal AC
Schulman BA
Komander D
Neefjes J
El Oualid F
Ovaa H
Source :
Nature chemical biology [Nat Chem Biol] 2016 Jul; Vol. 12 (7), pp. 523-30. Date of Electronic Publication: 2016 May 16.
Publication Year :
2016

Abstract

Post-translational modifications of proteins with ubiquitin (Ub) and ubiquitin-like modifiers (Ubls), orchestrated by a cascade of specialized E1, E2 and E3 enzymes, control a wide range of cellular processes. To monitor catalysis along these complex reaction pathways, we developed a cascading activity-based probe, UbDha. Similarly to the native Ub, upon ATP-dependent activation by the E1, UbDha can travel downstream to the E2 (and subsequently E3) enzymes through sequential trans-thioesterifications. Unlike the native Ub, at each step along the cascade, UbDha has the option to react irreversibly with active site cysteine residues of target enzymes, thus enabling their detection. We show that our cascading probe 'hops' and 'traps' catalytically active Ub-modifying enzymes (but not their substrates) by a mechanism diversifiable to Ubls. Our founder methodology, amenable to structural studies, proteome-wide profiling and monitoring of enzymatic activity in living cells, presents novel and versatile tools to interrogate Ub and Ubl cascades.

Details

Language :
English
ISSN :
1552-4469
Volume :
12
Issue :
7
Database :
MEDLINE
Journal :
Nature chemical biology
Publication Type :
Academic Journal
Accession number :
27182664
Full Text :
https://doi.org/10.1038/nchembio.2084