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

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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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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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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