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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.

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Figure 1: Mechanism of the ABP UbDha.
Figure 2: Covalent thioether bond formation of UbDha with conjugating enzymes.
Figure 3: Structural studies of thioether-linked E2–Ub adducts.
Figure 4: Proteome-wide activity profiling of Ub-conjugation machinery.
Figure 5: Activation of UbDha in vivo.
Figure 6: Probing in vivo E1–E2 cascade with UbDha.

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Acknowledgements

We thank members of H.O.'s lab for helpful discussion and reagents, J. Brown and S. Armour (Ubiquigent) for providing the E2 scan kit and D. El Atmioui for solid phase peptide synthesis. 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.

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Contributions

M.P.C.M. and F.E.O. designed the study. M.P.C.M., K.W. and I.B. carried out all labeling experiments. I.B. and K.W. designed and executed in-cell labeling experiments with assistance from R.M., and I.B. collected and analyzed confocal microscopy data. Mass spectrometry and relevant data analysis were performed by J.-G.C. and A.C.O.V. on samples prepared by K.W. and I.B. J.N.P. and D.K. performed structural and competition studies and analyzed NMR and X-ray data. K.-P.W. and B.A.S. generated the panel of purified HECT and NEDD8 pathway enzymes and helped with data analysis. J.N. helped with data analysis and provided helpful discussions. J.B. and M.G. provided UBA6. M.P.C.M., F.E.O. and H.O. managed the study. M.P.C.M. and I.B. wrote the manuscript with input from other authors.

Corresponding authors

Correspondence to Farid El Oualid or Huib Ovaa.

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Competing interests

H.O., M.P.C.M. and F.E.O. are entitled to royalties that may result from licensing patent application WO2016/032332 according to IP policies of the Netherlands Cancer Institute. H.O. and F.E.O. declare competing financial interests as cofounders and shareholders of UbiQ Bio BV. D.K. and H.O. are part of the DUB Alliance, which includes Cancer Research Technology and FORMA Therapeutics. D.K. is a consultant for FORMA Therapeutics.

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Supplementary Results, Supplementary Figures 1–22 and Supplementary Table 1 (PDF 2341 kb)

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Synthetic procedures (PDF 838 kb)

Supplementary Note 2

E1–E2–E3 labelling assay conditions (PDF 144 kb)

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Mulder, M., Witting, K., Berlin, I. et al. A cascading activity-based probe sequentially targets E1–E2–E3 ubiquitin enzymes. Nat Chem Biol 12, 523–530 (2016). https://doi.org/10.1038/nchembio.2084

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