Abstract

E3 ligases represent an important class of enzymes, yet there are currently no chemical probes for profiling their activity. We develop a new class of activity-based probe by re-engineering a ubiquitin-charged E2 conjugating enzyme and demonstrate the utility of these probes by profiling the transthiolation activity of the RING-in-between-RING (RBR) E3 ligase parkin in vitro and in cellular extracts. Our study provides valuable insight into the roles, and cellular hierarchy, of distinct phosphorylation events in parkin activation. We also profile parkin mutations associated with patients with Parkinson's disease and demonstrate that they mediate their effect largely by altering transthiolation activity. Furthermore, our probes enable direct and quantitative measurement of endogenous parkin activity, revealing that endogenous parkin is activated in neuronal cell lines (≥75%) in response to mitochondrial depolarization. This new technology also holds promise as a novel biomarker of PINK1-parkin signaling, as demonstrated by its compatibility with samples derived from individuals with Parkinson's disease.

  • Compound

    propargyl (E)-3-tosylacrylate

  • Compound

    (E)-N-propargyl-3-tosylacrylamide

  • Compound

    propargyl acrylate

  • Compound

    N-propargyl acrylamide

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Acknowledgements

We are grateful to the Medical Research Council Protein Phosphorylation and Ubiquitylation Unit Proteomics and DNA Sequencing Facilities. We are also grateful to I. Gilbert for support with chemistry instrumentation and access to modeling software. We also thank J.W. Chin (Medical Research Council Laboratory of Molecular Biology) for critical reading of the manuscript. This work was funded by the Scottish Funding Council, the UK Medical Research Council (MC_UU_12016/8) and pharmaceutical companies supporting the Division of Signal Transduction Therapy (AstraZeneca, Boehringer-Ingelheim, GlaxoSmithKline, Merck KGaA, Janssen Pharmaceutica and Pfizer). K.B. is funded by an A.J. Macdonald Menzies Charitable Trust Prize Studentship. M.M.K.M. is funded by a Wellcome Trust Senior Research Fellowship in Clinical Science (101022/Z/13/Z).

Author information

Affiliations

  1. Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, UK.

    • Kuan-Chuan Pao
    • , Mathew Stanley
    • , Cong Han
    • , Yu-Chiang Lai
    • , Paul Murphy
    • , Kristin Balk
    • , Nicola T Wood
    • , Miratul M K Muqit
    •  & Satpal Virdee
  2. Sorbonne Universités, Université Pierre et Marie Curie; Institut National de la Santé et de la Recherche Médicale, Unité 1127, Centre d'investigation Clinique 1422; Centre National de la Recherche Scientifique, Unité 7225; Assistance Publique Hôpitaux de Paris; Institut du Cerveau et de la Moelle, Paris, France.

    • Olga Corti
    •  & Jean-Christophe Corvol
  3. School of Medicine, University of Dundee, Dundee, UK.

    • Miratul M K Muqit

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Contributions

S.V. conceived the project. Experiments were designed by S.V., M.M.K.M., K.-C.P. and M.S. K.-C.P. carried out experiments with assistance from M.S., C.H., Y.-C.L, P.M., K.B. and S.V. M.S. also carried out small-molecule synthesis. N.T.W. performed cloning. J.-C.C. and O.C. generated and provided fibroblasts derived from patients with PD. S.V. and M.M.K.M. wrote the manuscript with input from other authors.

Competing interests

S.V., K.-C.P. and M.S. are authors on a patent that has been filed (PCT/GB2015/052860) relating to work presented in this article.

Corresponding author

Correspondence to Satpal Virdee.

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DOI

https://doi.org/10.1038/nchembio.2045

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