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Dynamic imaging of protease activity with fluorescently quenched activity-based probes

Nature Chemical Biology volume 1, pages 203–209 (2005)Cite this article

Abstract

Protease activity is tightly regulated in both normal and disease conditions. However, it is often difficult to monitor the dynamic nature of this regulation in the context of a live cell or whole organism. To address this limitation, we developed a series of quenched activity-based probes (qABPs) that become fluorescent upon activity-dependent covalent modification of a protease target. These reagents freely penetrate cells and allow direct imaging of protease activity in living cells. Targeted proteases are directly identified and monitored biochemically by virtue of the resulting covalent tag, thereby allowing unambiguous assignment of protease activities observed in imaging studies. We report here the design and synthesis of a selective, cell-permeable qABP for the study of papain-family cysteine proteases. This probe is used to monitor real-time protease activity in live human cells with fluorescence microscopy techniques as well as standard biochemical methods.

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Figure 1: Design of a qABP.
Figure 2: Labeling of recombinant cathepsins and intact cells with the control ABP and qABP.
Figure 3: Imaging protease activity in live cells.
Figure 4: Imaging protease activity in three-dimensional cultured cells.

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Acknowledgements

We thank B. Goulet and A. Nepveu (McGill University) for cathepsin L–deficient MEF cells and V. Turk and B. Turk (J. Stefan Institute) for recombinant human cathepsin L. The authors thank C. Gilon for helpful advice on peptide synthesis, G. von Degenfeld for technical assistance throughout the project, K. Boatright and S. Verhelst for helpful discussion of the manuscript. This work was supported by a Turman Fellowship at Stanford University (to M.B.), a US National Institutes of Health National Technology Center for Networks and Pathways grant U54 RR020843 (to M.B.), and a Department of Defense Breast Cancer Center of Excellence grant DAMD-17-02-0693 (to B.F.S.; M.B. subcontract).

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Authors and Affiliations

  1. Department of Pathology, Stanford University School of Medicine, 300 Pasteur Dr., Stanford, 94305, California, USA

    Galia Blum, Marko Fonovič, Mark J Rice & Matthew Bogyo

  2. Department of Pharmacology, Wayne State University School of Medicine, 540 East Canfield, Detroit, 48201, Michigan, USA

    Stefanie R Mullins, Christopher Jedeszko & Bonnie F Sloane

  3. Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine, 540 East Canfield, Detroit, 48201, Michigan, USA

    Stefanie R Mullins & Bonnie F Sloane

  4. Department of Biochemistry, Stanford University School of Medicine, 300 Pasteur Dr., Stanford, 94305, California, USA

    Kinneret Keren

  5. Department of Biochemistry and Molecular Biology, Jozef Stefan Institute, Jamova 39, Ljubljana, 1000, Slovenia

    Marko Fonovič

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Correspondence to Matthew Bogyo.

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Departments of Microbiology and Immunology, Stanford University School of Medicine, 300 Pasteur Dr., Stanford, 94305, California, USA

Supplementary information

Supplementary Fig. 1

Determination of quenching efficiency of the qABP GB117 relative to the unquenched control ABP GB111. (PDF 530 kb)

Supplementary Fig. 2

GB111 and GB117 where docked into cathepsin B (1SP4) and cathepsin L (1MHW) using the MMF94 force field using MOE (Chemical Computing Group). (PDF 3430 kb)

Supplementary Table 1

Inhibition rate constants of various probes for human cathepsin L and bovine cathepsin B. (PDF 35 kb)

Supplementary Methods (PDF 1983 kb)

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Blum, G., Mullins, S., Keren, K. et al. Dynamic imaging of protease activity with fluorescently quenched activity-based probes. Nat Chem Biol 1, 203–209 (2005). https://doi.org/10.1038/nchembio728

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