Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Probes of ubiquitin E3 ligases enable systematic dissection of parkin activation

Nature Chemical Biology volume 12, pages 324–331 (2016)Cite this article

Subjects

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Synthesis of alkyne-functionalized TDAEs and their utility for assembling E2∼Ub-based probes for profiling E3 transthiolation activity.
Figure 2: E2∼Ub-based probes label the RBR E3 ligase parkin in an activity-dependant manner.
Figure 3: Sustained parkin transthiolation activity requires phosphorylation of p-parkin and p-Ub and parkin.
Figure 4: Quantitative and direct activity-based protein profiling of transthiolation activity of parkin mutations found in patients with PD.
Figure 5: Activity-based profiling of cellular parkin provides insights into the hierachy of parkin and Ub phosphorylation in the context of PINK1-parkin signaling and reveals quantifiable, protonophore responsive activation of endogenous parkin in SH-SY5Y dopaminergic cells.
Figure 6: Activity-based profiling of PD patient–derived fibroblasts reveals biomarker potential.

Similar content being viewed by others

Accession codes

Accessions

Protein Data Bank

References

  1. Popovic, D., Vucic, D. & Dikic, I. Ubiquitination in disease pathogenesis and treatment. Nat. Med. 20, 1242–1253 (2014).

    CAS  PubMed  Google Scholar 

  2. Hershko, A. & Ciechanover, A. The ubiquitin system. Annu. Rev. Biochem. 67, 425–479 (1998).

    Article  CAS  PubMed  Google Scholar 

  3. Berndsen, C.E. & Wolberger, C. New insights into ubiquitin E3 ligase mechanism. Nat. Struct. Mol. Biol. 21, 301–307 (2014).

    CAS  PubMed  Google Scholar 

  4. Kitada, T. et al. Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism. Nature 392, 605–608 (1998).

    CAS  PubMed  Google Scholar 

  5. Wenzel, D.M., Lissounov, A., Brzovic, P.S. & Klevit, R.E. UBCH7 reactivity profile reveals parkin and HHARI to be RING/HECT hybrids. Nature 474, 105–108 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Pickrell, A.M. & Youle, R.J. The roles of PINK1, parkin, and mitochondrial fidelity in Parkinson's disease. Neuron 85, 257–273 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Chaugule, V.K. et al. Autoregulation of parkin activity through its ubiquitin-like domain. EMBO J. 30, 2853–2867 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Trempe, J.F. et al. Structure of parkin reveals mechanisms for ubiquitin ligase activation. Science 340, 1451–1455 (2013).

    CAS  PubMed  Google Scholar 

  9. Riley, B.E. et al. Structure and function of parkin E3 ubiquitin ligase reveals aspects of RING and HECT ligases. Nat. Commun. 4, 1982 (2013).

    CAS  PubMed  Google Scholar 

  10. Wauer, T. & Komander, D. Structure of the human parkin ligase domain in an autoinhibited state. EMBO J. 32, 2099–2112 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Kazlauskaite, A. et al. Parkin is activated by PINK1-dependent phosphorylation of ubiquitin at Ser65. Biochem. J. 460, 127–139 (2014).

    CAS  PubMed  Google Scholar 

  12. Kazlauskaite, A. et al. Phosphorylation of parkin at Serine65 is essential for activation: elaboration of a Miro1 substrate-based assay of parkin E3 ligase activity. Open Biol. 4, 130213 (2014).

    PubMed  PubMed Central  Google Scholar 

  13. Kane, L.A. et al. PINK1 phosphorylates ubiquitin to activate parkin E3 ubiquitin ligase activity. J. Cell Biol. 205, 143–153 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Koyano, F. et al. Ubiquitin is phosphorylated by PINK1 to activate parkin. Nature 510, 162–166 (2014).

    CAS  PubMed  Google Scholar 

  15. Kondapalli, C. et al. PINK1 is activated by mitochondrial membrane potential depolarization and stimulates parkin E3 ligase activity by phosphorylating Serine 65. Open Biol. 2, 120080 (2012).

    PubMed  PubMed Central  Google Scholar 

  16. Shiba-Fukushima, K. et al. PINK1-mediated phosphorylation of the parkin ubiquitin-like domain primes mitochondrial translocation of parkin and regulates mitophagy. Sci. Rep. 2, 1002 (2012).

    PubMed  PubMed Central  Google Scholar 

  17. Ordureau, A. et al. Quantitative proteomics reveal a feedforward mechanism for mitochondrial PARKIN translocation and ubiquitin chain synthesis. Mol. Cell 56, 360–375 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Ordureau, A. et al. Defining roles of PARKIN and ubiquitin phosphorylation by PINK1 in mitochondrial quality control using a ubiquitin replacement strategy. Proc. Natl. Acad. Sci. USA 112, 6637–6642 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Okatsu, K. et al. Phosphorylated ubiquitin chain is the genuine parkin receptor. J. Cell Biol. 209, 111–128 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Kazlauskaite, A. et al. Binding to serine 65-phosphorylated ubiquitin primes parkin for optimal PINK1-dependent phosphorylation and activation. EMBO Rep. 16, 939–954 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Wauer, T., Simicek, M., Schubert, A. & Komander, D. Mechanism of phospho-ubiquitin-induced PARKIN activation. Nature 524, 370–374 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Kazlauskaite, A. & Muqit, M.M. PINK1 and parkin – mitochondrial interplay between phosphorylation and ubiquitylation in parkinson's disease. FEBS J. 282, 215–223 (2015).

    CAS  PubMed  Google Scholar 

  23. Lazarou, M. et al. PINK1 drives parkin self-association and HECT-like E3 activity upstream of mitochondrial binding. J. Cell Biol. 200, 163–172 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Matsuda, N. et al. PINK1 stabilized by mitochondrial depolarization recruits parkin to damaged mitochondria and activates latent parkin for mitophagy. J. Cell Biol. 189, 211–221 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Cravatt, B.F., Wright, A.T. & Kozarich, J.W. Activity-based protein profiling: from enzyme chemistry to proteomic chemistry. Annu. Rev. Biochem. 77, 383–414 (2008).

    CAS  PubMed  Google Scholar 

  26. Stanley, M. et al. Orthogonal thiol functionalization at a single atomic center for profiling transthiolation activity of E1 activating enzymes. ACS Chem. Biol. 10, 1542–1554 (2015).

    CAS  PubMed  Google Scholar 

  27. Borodovsky, A. et al. A novel active site-directed probe specific for deubiquitylating enzymes reveals proteasome association of USP14. EMBO J. 20, 5187–5196 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Love, K.R., Pandya, R.K., Spooner, E. & Ploegh, H.L. Ubiquitin C-terminal electrophiles are activity-based probes for identification and mechanistic study of ubiquitin conjugating machinery. ACS Chem. Biol. 4, 275–287 (2009).

    PubMed  PubMed Central  Google Scholar 

  29. Smit, J.J. & Sixma, T.K. RBR E3-ligases at work. EMBO Rep. 15, 142–154 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Komander, D., Clague, M.J. & Urbé, S. Breaking the chains: structure and function of the deubiquitinases. Nat. Rev. Mol. Cell Biol. 10, 550–563 (2009).

    CAS  PubMed  Google Scholar 

  31. Olsen, S.K., Capili, A.D., Lu, X., Tan, D.S. & Lima, C.D. Active site remodelling accompanies thioester bond formation in the SUMO E1. Nature 463, 906–912 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. McGouran, J.F., Gaertner, S.R., Altun, M., Kramer, H.B. & Kessler, B.M. Deubiquitinating enzyme specificity for ubiquitin chain topology profiled by di-ubiquitin activity probes. Chem. Biol. 20, 1447–1455 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Mulder, M.P., El Oualid, F., ter Beek, J. & Ovaa, H. A native chemical ligation handle that enables the synthesis of advanced activity-based probes: diubiquitin as a case study. ChemBioChem 15, 946–949 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Haj-Yahya, N. et al. Dehydroalanine-based diubiquitin activity probes. Org. Lett. 16, 540–543 (2014).

    CAS  PubMed  Google Scholar 

  35. Li, G., Liang, Q., Gong, P., Tencer, A.H. & Zhuang, Z. Activity-based diubiquitin probes for elucidating the linkage specificity of deubiquitinating enzymes. Chem. Commun. (Camb.) 50, 216–218 (2014).

    CAS  Google Scholar 

  36. Borodovsky, A. et al. Chemistry-based functional proteomics reveals novel members of the deubiquitinating enzyme family. Chem. Biol. 9, 1149–1159 (2002).

    CAS  PubMed  Google Scholar 

  37. Niphakis, M.J. & Cravatt, B.F. Enzyme inhibitor discovery by activity-based protein profiling. Annu. Rev. Biochem. 83, 341–377 (2014).

    CAS  PubMed  Google Scholar 

  38. Nguyen, D.P. et al. Genetic encoding and labeling of aliphatic azides and alkynes in recombinant proteins via a pyrrolysyl-tRNA Synthetase/tRNA(CUA) pair and click chemistry. J. Am. Chem. Soc. 131, 8720–8721 (2009).

    CAS  PubMed  Google Scholar 

  39. Weissman, A.M. Themes and variations on ubiquitylation. Nat. Rev. Mol. Cell Biol. 2, 169–178 (2001).

    CAS  PubMed  Google Scholar 

  40. Lechtenberg, B.C. et al. Structure of a HOIP/E2∼ubiquitin complex reveals RBR E3 ligase mechanism and regulation. Nature 529, 546–550 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Sarraf, S.A. et al. Landscape of the PARKIN-dependent ubiquitylome in response to mitochondrial depolarization. Nature 496, 372–376 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Kamadurai, H.B. et al. Insights into ubiquitin transfer cascades from a structure of a UbcH5B∼ubiquitin-HECT(NEDD4L) complex. Mol. Cell 36, 1095–1102 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Stieglitz, B., Morris-Davies, A.C., Koliopoulos, M.G., Christodoulou, E. & Rittinger, K. LUBAC synthesizes linear ubiquitin chains via a thioester intermediate. EMBO Rep. 13, 840–846 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Lin, D.Y., Diao, J., Zhou, D. & Chen, J. Biochemical and structural studies of a HECT-like ubiquitin ligase from Escherichia coli O157:H7. J. Biol. Chem. 286, 441–449 (2011).

    CAS  PubMed  Google Scholar 

  45. Ekkebus, R. et al. On terminal alkynes that can react with active-site cysteine nucleophiles in proteases. J. Am. Chem. Soc. 135, 2867–2870 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Lazarou, M. et al. The ubiquitin kinase PINK1 recruits autophagy receptors to induce mitophagy. Nature 524, 309–314 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Kumar, A. et al. Disruption of the autoinhibited state primes the E3 ligase parkin for activation and catalysis. EMBO J. 34, 2506–2521 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Sauvé, V. et al. A Ubl/ubiquitin switch in the activation of parkin. EMBO J. 34, 2492–2505 (2015).

    PubMed  PubMed Central  Google Scholar 

  49. Okatsu, K. et al. A dimeric PINK1-containing complex on depolarized mitochondria stimulates parkin recruitment. J. Biol. Chem. 288, 36372–36384 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Lai, Y.C. et al. Phosphoproteomic screening identifies Rab GTPases as novel downstream targets of PINK1. EMBO J. 34, 2840–2861 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Katrun, P. & Chiampanichayakul, S.PhI. (OAc)2/KI-mediated reaction of aryl sulfinates with alkenes, alkynes, and α,β-unsaturated carbonyl compounds: synthesis of vinyl sulfones and β-iodovinyl sulfones. Eur. J. Org. Chem. 29, 5633–5641 (2010).

    Google Scholar 

  52. Dadová, J. et al. Vinylsulfonamide and acrylamide modification of DNA for cross-linking with proteins. Angew. Chem. Int. Edn. Engl. 52, 10515–10518 (2013).

    Google Scholar 

  53. Virdee, S., Ye, Y., Nguyen, D.P., Komander, D. & Chin, J.W. Engineered diubiquitin synthesis reveals Lys29-isopeptide specificity of an OTU deubiquitinase. Nat. Chem. Biol. 6, 750–757 (2010).

    CAS  PubMed  Google Scholar 

  54. Cui, C., Zhao, W., Chen, J., Wang, J. & Li, Q. Elimination of in vivo cleavage between target protein and intein in the intein-mediated protein purification systems. Protein Expr. Purif. 50, 74–81 (2006).

    CAS  PubMed  Google Scholar 

  55. El Oualid, F. et al. Chemical synthesis of ubiquitin, ubiquitin-based probes, and diubiquitin. Angew. Chem. Int. Edn. Engl. 49, 10149–10153 (2010).

    CAS  Google Scholar 

  56. Hong, V., Presolski, S.I., Ma, C. & Finn, M.G. Analysis and optimization of copper-catalyzed azide-alkyne cycloaddition for bioconjugation. Angew. Chem. Int. Edn. Engl. 48, 9879–9883 (2009).

    CAS  Google Scholar 

  57. Plechanovová, A., Jaffray, E.G., Tatham, M.H., Naismith, J.H. & Hay, R.T. Structure of a RING E3 ligase and ubiquitin-loaded E2 primed for catalysis. Nature 489, 115–120 (2012).

    PubMed  PubMed Central  Google Scholar 

  58. Sommer, S., Weikart, N.D., Linne, U. & Mootz, H.D. Covalent inhibition of SUMO and ubiquitin-specific cysteine proteases by an in situ thiol-alkyne addition. Bioorg. Med. Chem. 21, 2511–2517 (2013).

    CAS  PubMed  Google Scholar 

  59. Yang, B. et al. Identification of cross-linked peptides from complex samples. Nat. Methods 9, 904–906 (2012).

    CAS  PubMed  Google Scholar 

  60. Narendra, D.P. et al. PINK1 is selectively stabilized on impaired mitochondria to activate parkin. PLoS Biol. 8, e1000298 (2010).

    PubMed  PubMed Central  Google Scholar 

Download references

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

Authors and 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,

    Olga Corti & Jean-Christophe Corvol

  3. Centre National de la Recherche Scientifique, Unité 7225,

    Olga Corti & Jean-Christophe Corvol

  4. Assistance Publique Hôpitaux de Paris; Institut du Cerveau et de la Moelle,

    Olga Corti & Jean-Christophe Corvol

  5. Assistance Publique Hôpitaux de Paris,

    Olga Corti & Jean-Christophe Corvol

  6. Institut du Cerveau et de la Moelle,

    Olga Corti & Jean-Christophe Corvol

  7. Institut National de la Santé et de la Recherche Médicale, Unité 1127, Centre d'investigation Clinique 1422, Paris, France

    Olga Corti & Jean-Christophe Corvol

  8. School of Medicine, University of Dundee, Dundee, UK

    Miratul M K Muqit

Authors
  1. Kuan-Chuan Pao
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mathew Stanley
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Cong Han
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yu-Chiang Lai
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Paul Murphy
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Kristin Balk
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Nicola T Wood
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Olga Corti
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Jean-Christophe Corvol
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Miratul M K Muqit
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Satpal Virdee
    View author publications

    You can also search for this author in PubMed Google Scholar

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.

Corresponding author

Correspondence to Satpal Virdee.

Ethics declarations

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.

Supplementary information

Supplementary Text and Figures

Supplementary Results and Supplementary Figures 1–22. (PDF 10033 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Pao, KC., Stanley, M., Han, C. et al. Probes of ubiquitin E3 ligases enable systematic dissection of parkin activation. Nat Chem Biol 12, 324–331 (2016). https://doi.org/10.1038/nchembio.2045

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

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

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing