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

Monique P.C. Mulder, Katharina Witting, Ilana Berlin, Jonathan Pruneda, Kuen Phon Wu, Jer Gung Chang, Remco Merkx, Johanna Bialas, Marcus Groettrup, Alfred C.O. Vertegaal, Brenda A. Schulman, David Komander, Jacques Neefjes, Farid El Oualid, Huib Ovaa

Research output: Contribution to journalArticle

37 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)523-530
Number of pages8
JournalNature Chemical Biology
Volume12
Issue number7
DOIs
StatePublished - Jul 1 2016
Externally publishedYes

Fingerprint

Ubiquitin
Enzymes
Humulus
Proteome
Post Translational Protein Processing
Catalysis
Cysteine
Catalytic Domain
Adenosine Triphosphate

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

Cite this

Mulder, M. P. C., Witting, K., Berlin, I., Pruneda, J., Wu, K. P., Chang, J. G., ... Ovaa, H. (2016). A cascading activity-based probe sequentially targets E1-E2-E3 ubiquitin enzymes. Nature Chemical Biology, 12(7), 523-530. https://doi.org/10.1038/nchembio.2084

A cascading activity-based probe sequentially targets E1-E2-E3 ubiquitin enzymes. / Mulder, Monique P.C.; Witting, Katharina; Berlin, Ilana; Pruneda, Jonathan; Wu, Kuen Phon; Chang, Jer Gung; Merkx, Remco; Bialas, Johanna; Groettrup, Marcus; Vertegaal, Alfred C.O.; Schulman, Brenda A.; Komander, David; Neefjes, Jacques; El Oualid, Farid; Ovaa, Huib.

In: Nature Chemical Biology, Vol. 12, No. 7, 01.07.2016, p. 523-530.

Research output: Contribution to journalArticle

Mulder, MPC, Witting, K, Berlin, I, Pruneda, J, Wu, KP, Chang, JG, Merkx, R, Bialas, J, Groettrup, M, Vertegaal, ACO, Schulman, BA, Komander, D, Neefjes, J, El Oualid, F & Ovaa, H 2016, 'A cascading activity-based probe sequentially targets E1-E2-E3 ubiquitin enzymes', Nature Chemical Biology, vol. 12, no. 7, pp. 523-530. https://doi.org/10.1038/nchembio.2084
Mulder, Monique P.C. ; Witting, Katharina ; Berlin, Ilana ; Pruneda, Jonathan ; Wu, Kuen Phon ; Chang, Jer Gung ; Merkx, Remco ; Bialas, Johanna ; Groettrup, Marcus ; Vertegaal, Alfred C.O. ; Schulman, Brenda A. ; Komander, David ; Neefjes, Jacques ; El Oualid, Farid ; Ovaa, Huib. / A cascading activity-based probe sequentially targets E1-E2-E3 ubiquitin enzymes. In: Nature Chemical Biology. 2016 ; Vol. 12, No. 7. pp. 523-530.
@article{fb89142542094a0d98941ad8a800146f,
title = "A cascading activity-based probe sequentially targets E1-E2-E3 ubiquitin enzymes",
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.",
author = "Mulder, {Monique P.C.} and Katharina Witting and Ilana Berlin and Jonathan Pruneda and Wu, {Kuen Phon} and Chang, {Jer Gung} and Remco Merkx and Johanna Bialas and Marcus Groettrup and Vertegaal, {Alfred C.O.} and Schulman, {Brenda A.} and David Komander and Jacques Neefjes and {El Oualid}, Farid and Huib Ovaa",
year = "2016",
month = "7",
day = "1",
doi = "10.1038/nchembio.2084",
language = "English (US)",
volume = "12",
pages = "523--530",
journal = "Nature Chemical Biology",
issn = "1552-4450",
publisher = "Nature Publishing Group",
number = "7",

}

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

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

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

VL - 12

SP - 523

EP - 530

JO - Nature Chemical Biology

JF - Nature Chemical Biology

SN - 1552-4450

IS - 7

ER -