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β-Lactone probes identify a papain-like peptide ligase in Arabidopsis thaliana

Nature Chemical Biology volume 4, pages 557–563 (2008)Cite this article

Abstract

New activity-based probes are essential for expanding studies on the hundreds of serine and cysteine proteases encoded by the genome of Arabidopsis thaliana. To monitor protease activities in plant extracts, we generated biotinylated peptides containing a β-lactone reactive group. These probes cause strong labeling in leaf proteomes. Unexpectedly, labeling was detected at the N terminus of PsbP, nonproteolytic protein of photosystem II. Inhibitor studies and reverse genetics led to the discovery that this unusual modification is mediated by a single plant-specific, papain-like protease called RD21. In cellular extracts, RD21 accepts both β-lactone probes and peptides as donor molecules and ligates them, probably through a thioester intermediate, to unmodified N termini of acceptor proteins.

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Figure 1: β-lactone probes and their labeling of leaf extracts.
Figure 2: Identification of the major IS4-labeled protein.
Figure 3: IS4 labeling requires cysteine protease RD21.
Figure 4: Binding of β-lactones to RD21.
Figure 5: RD21 can ligate peptides.
Figure 6: Model for β-lactone and peptide labeling of PsbP by RD21.

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References

  1. Beers, E.P., Jones, A.M. & Dickerman, A.W. The S8 serine, C1A cysteine and A1 aspartic protease families in Arabidopsis. Phytochemistry 65, 43–58 (2004).

    Article  CAS  Google Scholar 

  2. Van der Hoorn, R.A.L. Plant proteases: from phenotypes to molecular mechanisms. Annu. Rev. Plant Biol. 59, 191–223 (2008).

    Article  CAS  Google Scholar 

  3. Clemens, S. Evolution and function of phytochelatin synthases. J. Plant Physiol. 163, 319–332 (2006).

    Article  CAS  Google Scholar 

  4. Lehfeldt, C. et al. Cloning of the SNG1 gene of Arabidopsis reveals a role for a serine carboxypeptidase-like protein as an acyltransferase in secondary metabolism. Plant Cell 12, 1295–1306 (2000).

    Article  CAS  Google Scholar 

  5. Evans, M.J. & Cravatt, B.F. Mechanism-based profiling of enzyme families. Chem. Rev. 106, 3279–3301 (2006).

    Article  CAS  Google Scholar 

  6. Fonovic, M. & Bogyo, M. Activity-based probes for proteases: application to biomarker discovery, molecular imaging and drug screening. Curr. Pharm. Des. 13, 253–261 (2007).

    Article  CAS  Google Scholar 

  7. Greenbaum, D., Medzihradszky, K.F., Burlingame, A. & Bogyo, M. Epoxide electrophiles as activity-dependent cysteine protease profiling and discovery tools. Chem. Biol. 7, 569–581 (2000).

    Article  CAS  Google Scholar 

  8. Kato, D. et al. Activity-based probes that target diverse cysteine protease families. Nat. Chem. Biol. 1, 33–38 (2005).

    Article  CAS  Google Scholar 

  9. Hemelaar, J. et al. Specific and covalent targeting of conjugating and deconjugating enzymes of ubiquitin-like proteins. Mol. Cell. Biol. 24, 84–95 (2004).

    Article  CAS  Google Scholar 

  10. Mahrus, S. & Craik, C.S. Selective chemical functional probes of granzymes A and B reveal granzyme B is a major effector of natural killer cell-mediated lysis of target cells. Chem. Biol. 12, 567–577 (2005).

    Article  CAS  Google Scholar 

  11. Pan, Z. et al. Development of activity-based probes for trypsin-family serine proteases. Bioorg. Med. Chem. Lett. 16, 2882–2885 (2006).

    Article  CAS  Google Scholar 

  12. Liu, Y., Patricelli, M.P. & Cravatt, B.F. Activity-based protein profiling: the serine hydrolases. Proc. Natl. Acad. Sci. USA 96, 14694–14699 (1999).

    Article  CAS  Google Scholar 

  13. Van der Hoorn, R.A.L., Leeuwenburgh, M.A., Bogyo, M., Joosten, M.H.A.J. & Peck, S.C. Activity profiling of papain-like cysteine proteases in plants. Plant Physiol. 135, 1170–1178 (2004).

    Article  CAS  Google Scholar 

  14. Shabab, M. et al. Fungal effector protein AVR2 targets diversifying defence-related Cys proteases of tomato. Plant Cell 20, 1169–1183 (2008).

    Article  CAS  Google Scholar 

  15. Lall, M.S., Karvellas, C. & Vederas, J.C. β-Lactones as a new class of cysteine proteinase inhibitors: inhibition of hepatitis A virus 3C proteinase by N-Cbz-serine β-lactone. Org. Lett. 1, 803–806 (1999).

    Article  CAS  Google Scholar 

  16. Dick, L.R. et al. Mechanistic studies on the inactivation of the proteasome by lactacystin in cultured cells. J. Biol. Chem. 272, 182–188 (1997).

    Article  CAS  Google Scholar 

  17. Drahl, C., Cravatt, B.F. & Sorensen, E.J. Protein-reactive natural products. Angew. Chem. Int. Edn. 44, 5788–5809 (2005).

    Article  CAS  Google Scholar 

  18. Böttcher, T. & Sieber, S.A. β-lactones as privileged structures for the active-site labeling of versatile bacterial enzyme classes. Angew. Chem. Int. Edn. 47, 4600–4603 (2008).

    Article  Google Scholar 

  19. Yamada, K., Matsushima, R., Nishimura, M. & Hara-Nishimura, I. A slow maturation of a cysteine protease with a granulin domain in the vacuoles of senescing Arabidopsis leaves. Plant Physiol. 127, 1626–1634 (2001).

    Article  CAS  Google Scholar 

  20. Yi, X., Hargett, S.R., Liu, H., Frankel, L.K. & Bricker, T.M. The PsbP protein is required for photosystem II complex assembly/stability and photoautotrophy in Arabidopsis thaliana. J. Biol. Chem. 282, 24833–24841 (2007).

    Article  CAS  Google Scholar 

  21. Van der Hoorn, R.A.L., Laurent, F., Roth, R. & De Wit, P.J.G.M. Agroinfiltration is a versatile tool that facilitates comparative analysis of Avr9/Cf-9-induced and Avr4/Cf-4-induced necrosis. Mol. Plant Microbe Interact. 13, 439–446 (2000).

    Article  CAS  Google Scholar 

  22. Voinnet, O., Rivas, S., Mestre, P. & Baulcombe, D. An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus. Plant J. 33, 949–956 (2003).

    Article  CAS  Google Scholar 

  23. Powers, J.C., Asgian, J.L., Ekici, O.D. & James, K.E. Irreversible inhibitors of serine, cysteine, and threonine proteases. Chem. Rev. 102, 4639–4750 (2002).

    Article  CAS  Google Scholar 

  24. Bateman, A. & Bennett, H.P.J. Granulins: the structure and function of an emerging family of growth factors. J. Endocrinol. 158, 145–151 (1998).

    Article  CAS  Google Scholar 

  25. Walling, L.L. Recycling or regulation? The role of amino-terminal modifying enzymes. Curr. Opin. Plant Biol. 9, 227–233 (2006).

    Article  CAS  Google Scholar 

  26. Hayashi, Y. et al. A proteinase-storing body that prepares for cell death or stresses in the epidermal cells of Arabidopsis. Plant Cell Physiol. 42, 894–899 (2001).

    Article  CAS  Google Scholar 

  27. Carter, C. et al. The vegetative vacuole proteome of Arabidopsis thaliana reveals predicted and unexpected proteins. Plant Cell 16, 3285–3303 (2004).

    Article  CAS  Google Scholar 

  28. Tabaeizadeh, Z. et al. Identification and immunolocalization of a 65 kDa drought induced protein in cultivated tomato Lycopersicon esculentum. Protoplasma 186, 208–219 (1995).

    Article  CAS  Google Scholar 

  29. Cobbett, C. & Goldsbrough, P. Phytochelatins and metallothioneins: roles in heavy metal detoxification and homeostasis. Annu. Rev. Plant Biol. 53, 159–182 (2002).

    Article  CAS  Google Scholar 

  30. Saska, I. et al. An asparaginyl endopeptidase mediates in vivo protein backbone cyclization. J. Biol. Chem. 282, 28721–28728 (2007).

    Article  Google Scholar 

  31. Lombard, C., Saulnier, J. & Wallach, J.M. Recent trends in protease-catalyzed peptide synthesis. Protein Pept. Lett. 12, 621–629 (2005).

    Article  CAS  Google Scholar 

  32. Popp, M.W., Antos, J.M., Grotenberg, G.M., Spooner, E. & Ploegh, H.L. Sortagging: a versatile method for protein labeling. Nat. Chem. Biol. 3, 707–708 (2007).

    Article  CAS  Google Scholar 

  33. Tanaka, T., Yamamoto, T., Tsukiji, S. & Nagamune, T. Site-specific protein modification on living cells catalyzed by sortase. ChemBioChem 9, 802–807 (2008).

    Article  CAS  Google Scholar 

  34. Chang, T.K., Jackson, D.Y., Burnier, J.P. & Wells, J.A. Subtiligase: a tool for semisynthesis of proteins. Proc. Natl. Acad. Sci. USA 91, 12544–12548 (1994).

    Article  CAS  Google Scholar 

  35. Tan, X.H., Zhang, X., Yang, R. & Liu, C.F. A simple method for preparing peptide C-terminal thioacids and their application in sequential chemoenzymatic ligation. ChemBioChem 9, 1052–1056 (2008).

    Article  CAS  Google Scholar 

  36. Van der Hoorn, R.A.L. et al. Structure-function analysis of Cf-9, a receptor-like protein with extracytoplasmic leucine-rich repeats. Plant Cell 17, 1000–1015 (2005).

    Article  CAS  Google Scholar 

  37. Gobom, J. et al. Alpha-cyano-4-hydroxycinnamic acid affinity sample preparation. A protocol for MALDI-MS peptide analysis in proteomics. Anal. Chem. 73, 434–438 (2001).

    Article  CAS  Google Scholar 

  38. Suckau, D. et al. A novel MALDI LIFT-TOF/TOF mass spectrometer for proteomics. Anal. Bioanal. Chem. 376, 952–965 (2003).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank C. MacKintosh (University of Dundee) for providing the antibodies to RD21 and C. Koncz (Max Planck Institute for Plant Breeding Research) for providing the rd21B knockout. This work was supported by the International Max Planck Research School (to C.G. and T.S.), the Arabidopsis Functional Genomics Netwerk of the Deutsche Forschungsgemeinschaft (to T.C.), the Alexander von Humboldt Foundation (to R.B.) and the Max Planck Society (to Z.W., R.B., H.W., M.K. and R.A.L.v.d.H.).

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Author notes

  1. Zheming Wang and Christian Gu: These authors contributed equally to this work.

Authors and Affiliations

  1. Plant Chemetics Lab, Max Planck Institute for Plant Breeding Research, Carl-von-Linne Weg 10, Cologne, 50829, Germany

    Zheming Wang, Christian Gu, Takayuki Shindo & Renier A L van der Hoorn

  2. Chemical Genomics Centre of the Max Planck Society, Otto-Hahn-Straße 11, Dortmund, 44227, Germany

    Zheming Wang, Markus Kaiser & Renier A L van der Hoorn

  3. Proteomics Service Centre, Max Planck Institute for Plant Breeding Research, Carl-von-Linne Weg 10, Cologne, 50829, Germany

    Tom Colby

  4. Max Planck Institute for Molecular Physiology, Otto-Hahn-Straße 11, Dortmund, 44227, Germany

    Rengarajan Balamurugan & Herbert Waldmann

  5. School of Chemistry, University of Hyderabad, Gachi Bowli, Hyderabad, 500 046, India

    Rengarajan Balamurugan

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  1. Zheming Wang
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Correspondence to Renier A L van der Hoorn.

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Wang, Z., Gu, C., Colby, T. et al. β-Lactone probes identify a papain-like peptide ligase in Arabidopsis thaliana. Nat Chem Biol 4, 557–563 (2008). https://doi.org/10.1038/nchembio.104

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