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

Monique P.C. Mulder, Katharina Witting, Ilana Berlin, Jonathan N. 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

53 Scopus citations

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

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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    Mulder, M. P. C., Witting, K., Berlin, I., Pruneda, J. N., Wu, K. P., Chang, J. G., Merkx, R., Bialas, J., Groettrup, M., Vertegaal, A. C. O., Schulman, B. A., 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, 12(7), 523-530. https://doi.org/10.1038/nchembio.2084