Letter abstract
Nature Chemical Biology 1, 203 - 209 (2005)
Published online: 14 August 2005 | doi:10.1038/nchembio728
Dynamic imaging of protease activity with fluorescently quenched activity-based probes
Galia Blum1,
Stefanie R Mullins2,3,
Kinneret Keren4,
Marko Fonovi
1,5,
Christopher Jedeszko2,
Mark J Rice1,
Bonnie F Sloane2,3
&
Matthew Bogyo1
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.
- Department of Pathology, Stanford University School of Medicine, 300 Pasteur Dr., Stanford, California 94305, USA.
- Department of Pharmacology, Wayne State University School of Medicine, 540 East Canfield, Detroit, Michigan 48201, USA.
- Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine, 540 East Canfield, Detroit, Michigan 48201, USA.
- Department of Biochemistry, Stanford University School of Medicine, 300 Pasteur Dr., Stanford, California 94305, USA.
- Department of Biochemistry and Molecular Biology, Jozef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia.
- Departments of Microbiology and Immunology, Stanford University School of Medicine, 300 Pasteur Dr., Stanford, California 94305, USA.
Correspondence to: Matthew Bogyo1 e-mail: mbogyo@stanford.edu
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