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Activity-based probes that target diverse cysteine protease families

Nature Chemical Biology volume 1, pages 33–38 (2005)Cite this article

Abstract

Proteases are one of the largest and best-characterized families of enzymes in the human proteome. Unfortunately, the understanding of protease function in the context of complex proteolytic cascades remains in its infancy. One major reason for this gap in understanding is the lack of technologies that allow direct assessment of protease activity. We report here an optimized solid-phase synthesis protocol that allows rapid generation of activity-based probes (ABPs) targeting a range of cysteine protease families. These reagents selectively form covalent bonds with the active-site thiol of a cysteine protease, allowing direct biochemical profiling of protease activities in complex proteomes. We present a number of probes containing either a single amino acid or an extended peptide sequence that target caspases, legumains, gingipains and cathepsins. Biochemical studies using these reagents highlight their overall utility and provide insight into the biochemical functions of members of these protease families.

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Figure 1: Specificity of diverse P1 amino acid AOMK probes.
Figure 2: ABPs label endogenous legumain and reveal a pH switch that governs legumain specificity.
Figure 3: AOMK-based probes selectively target caspases in crude proteomes.
Figure 4: Optimization of cathepsin B–specific AOMK probes.

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Acknowledgements

We thank C. Watts (University of Dundee) for legumain antibodies, C. Peters and T. Reinheckel (Albert-Ludwigs-Universität Freiburg) for legumain-knockout mouse tissues, H. Chapman and J. Lee (University of California, San Francisco) for recombinant mouse legumain, V. Turk and B. Turk (J. Stefan Institute) for recombinant human cathepsin L, J. Potempa (University of Georgia) for P. gingivalis cell extracts and purified gingipains, and T. Burster (Stanford University) for cell lines. We thank A. Price and S. Snipas for technical assistance, L. Carpino (University of Massachusetts) for critical advice on the removal of the Fmoc protecting group and J. Ellman, A. Lee, L. Huang and W. Wood for helpful discussions. This work was supported by a Turman Fellowship at Stanford University (to M.B.), a National Institutes of Health National Technology Center for Networks and Pathways grant U54 RR020843 (to M.B. and G.S.S.) and a Department of Defense Breast Cancer Center of Excellence grant DAMD-17-02-0693 (to B. Sloane—M.B. subcontract). A.B. was funded by a National Human Genome Research Institute training grant 5T32 HG00044.

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  1. Daisuke Kato and Kelly M Boatright: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Pathology, Stanford University School of Medicine, 300 Pasteur Dr., Stanford, 940305, California, USA

    Daisuke Kato, Kelly M Boatright, Tamim Nazif, Galia Blum & Matthew Bogyo

  2. Cancer Biology Graduate Program, Stanford University School of Medicine, 300 Pasteur Dr., Stanford, 940305, California, USA

    Alicia B Berger

  3. The Burnham Institute, 10901 N. Torrey Pines Road, La Jolla, 92037, California, USA

    Ciara Ryan & Guy S Salvesen

  4. Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, 94143, California, USA

    Kareem A H Chehade & Matthew Bogyo

  5. Department of Microbiology and Immunology, Stanford University School of Medicine, 300 Pasteur Dr., Stanford, 940305, California, USA

    Matthew Bogyo

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Correspondence to Matthew Bogyo.

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Supplementary information

Supplementary Fig. 1

Optimization of Fmoc deprotection during solid phase synthesis of peptide AOMK probes. (PDF 776 kb)

Supplementary Fig. 2

Labeling of protease targets using simple P1 AOMK probes in crude proteomes. (PDF 3622 kb)

Supplementary Methods (PDF 4786 kb)

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Kato, D., Boatright, K., Berger, A. et al. Activity-based probes that target diverse cysteine protease families. Nat Chem Biol 1, 33–38 (2005). https://doi.org/10.1038/nchembio707

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