TY - JOUR
T1 - A cascading activity-based probe sequentially targets E1-E2-E3 ubiquitin enzymes
AU - Mulder, Monique P.C.
AU - Witting, Katharina
AU - Berlin, Ilana
AU - Pruneda, Jonathan N.
AU - Wu, Kuen Phon
AU - Chang, Jer Gung
AU - Merkx, Remco
AU - Bialas, Johanna
AU - Groettrup, Marcus
AU - Vertegaal, Alfred C.O.
AU - Schulman, Brenda A.
AU - Komander, David
AU - Neefjes, Jacques
AU - El Oualid, Farid
AU - Ovaa, Huib
N1 - Funding Information:
We acknowledge beamline staff at Diamond I04-1 for expert help. Work was supported by a VICI grant from the Netherlands Organization for Scientific Research (NWO) (724013002) to H.O., a Marie Curie ITN fellowship (290257) to K.W. and EMBO long-term fellowships to I.B. and J.N.P. Work in D.K.'s lab is funded by Medical Research Council (U105192732), the European Research Council (309756), and the Lister Institute for Preventive Medicine. Work in B.A.S.'s lab is funded by ALSAC, HHMI and NIH grant R37GM069530. Work in A.C.O.V.'s lab is funded by the NWO (93511037) and the European Research Council (310913). Work in M.G.'s lab is funded by the German Research Foundation (DFG) CRC969, project C01. J.B. received a stipend from the Graduate School Chemical Biology Ko-RSCB.
PY - 2016/7/1
Y1 - 2016/7/1
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=84968610510&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84968610510&partnerID=8YFLogxK
U2 - 10.1038/nchembio.2084
DO - 10.1038/nchembio.2084
M3 - Article
C2 - 27182664
AN - SCOPUS:84968610510
SN - 1552-4450
VL - 12
SP - 523
EP - 530
JO - Nature Chemical Biology
JF - Nature Chemical Biology
IS - 7
ER -