Dynamic imaging of protease activity with fluorescently quenched activity-based probes.
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.
Blum
Galia
G
Department of Pathology, Stanford University School of Medicine, 300 Pasteur Dr., Stanford, California 94305, USA.
Mullins
Stefanie R
SR
Keren
Kinneret
K
Fonovic
Marko
M
Jedeszko
Christopher
C
Rice
Mark J
MJ
Sloane
Bonnie F
BF
Bogyo
Matthew
M
D000818:Animals; D002403:Cathepsins / Q000737:chemistry / Q000235:genetics; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D051379:Mice; D015335:Molecular Probes / Q000737:chemistry* / Q000378:metabolism*; D015394:Molecular Structure; D041681:NIH 3T3 Cells; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
D005456:Fluorescent Dyes; D015335:Molecular Probes; D011994:Recombinant Proteins; D002403:Cathepsins; D003546:Cysteine Endopeptidases