BACKGROUND: A number of fluorescent caspase substrates and FRET-based indicators have been developed to study the in vivo activation of caspases, a conserved family of proteases critical in inflammatory, and apoptosis signaling pathways. To date, all substrates have measured only one caspase activity. Here, we describe a FRET-based probe for simultaneously measuring two distinct caspase activities in living cells. METHODS: This probe consists of a CFP-YFP-mRFP fusion protein containing a caspase-3-cleavage motif, DEVD, between CFP and YFP, and a caspase-6-cleavage site, VEID, between YFP and mRFP. DEVDase and VEIDase activities could be assessed simultaneously by monitoring diminished FRET mediated by cleavage of either or both of these protease cleavage sites using flow cytometry. RESULTS: DEVDase and VEIDase activities were completely inhibited by the pan-caspase inhibitor z-VAD-fmk and enhanced by DNA-damaging drugs or by anti-Fas stimulation. DEVD and VEID cleavage specificities were validated by using caspase-3-deficient MCF7-Fas cells and a caspase-6-specific inhibitor. Kinetic analysis with the FRET probe revealed that caspase-3 activation consistently preceded caspase-6 by approximately 30 min following induction of apoptosis. CONCLUSIONS: We have developed a novel FRET-based probe for simultaneous detection of two caspase activities in living cells using flow cytometry. Simultaneous detection of two caspase activities using this probe has clearly provided information of the ordering of caspase-3 and -6 in the apoptotic pathway. Copyright 2006 International Society for Analytical Cytology.
BACKGROUND: A number of fluorescent caspase substrates and FRET-based indicators have been developed to study the in vivo activation of caspases, a conserved family of proteases critical in inflammatory, and apoptosis signaling pathways. To date, all substrates have measured only one caspase activity. Here, we describe a FRET-based probe for simultaneously measuring two distinct caspase activities in living cells. METHODS: This probe consists of a CFP-YFP-mRFP fusion protein containing a caspase-3-cleavage motif, DEVD, between CFP and YFP, and a caspase-6-cleavage site, VEID, between YFP and mRFP. DEVDase and VEIDase activities could be assessed simultaneously by monitoring diminished FRET mediated by cleavage of either or both of these protease cleavage sites using flow cytometry. RESULTS: DEVDase and VEIDase activities were completely inhibited by the pan-caspase inhibitor z-VAD-fmk and enhanced by DNA-damaging drugs or by anti-Fas stimulation. DEVD and VEID cleavage specificities were validated by using caspase-3-deficient MCF7-Fas cells and a caspase-6-specific inhibitor. Kinetic analysis with the FRET probe revealed that caspase-3 activation consistently preceded caspase-6 by approximately 30 min following induction of apoptosis. CONCLUSIONS: We have developed a novel FRET-based probe for simultaneous detection of two caspase activities in living cells using flow cytometry. Simultaneous detection of two caspase activities using this probe has clearly provided information of the ordering of caspase-3 and -6 in the apoptotic pathway. Copyright 2006 International Society for Analytical Cytology.
Authors: Xinglu Huang; Magdalena Swierczewska; Ki Young Choi; Lei Zhu; Ashwinkumar Bhirde; Jinwoo Park; Kwangmeyung Kim; Jin Xie; Gang Niu; Kang Choon Lee; Seulki Lee; Xiaoyuan Chen Journal: Angew Chem Int Ed Engl Date: 2012-01-02 Impact factor: 15.336
Authors: Marcin Poreba; Aleksandra Szalek; Paulina Kasperkiewicz; Wioletta Rut; Guy S Salvesen; Marcin Drag Journal: Chem Rev Date: 2015-11-09 Impact factor: 60.622
Authors: Yu-Ran Na; So Yeon Kim; Jellert T Gaublomme; Alex K Shalek; Marsela Jorgolli; Hongkun Park; Eun Gyeong Yang Journal: Nano Lett Date: 2012-12-20 Impact factor: 11.189