Dynamic Imaging of Protease Activity With Fluorescently Quenched Activity-Based Probes

Galia Blum, Stefanie R. Mullins, Kinneret Keren, Marko Fonovič, Christopher Jedeszko, Mark J. Rice, Bonnie F. Sloane, Matthew Bogyo*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

308 Scopus citations

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.

Original languageAmerican English
Pages (from-to)203-209
Number of pages7
JournalNature Chemical Biology
Volume1
Issue number4
DOIs
StatePublished - Aug 2005
Externally publishedYes

Bibliographical note

Funding Information:
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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