Novel mutant green fluorescent protein protease substrates reveal the activation of specific caspases during apoptosis.
BACKGROUND
The caspase-mediated proteolysis of many cellular proteins is a critical event during programmed cell death or apoptosis. It is important to determine which caspases are activated in mammalian cells, and where and when activation occurs, upon receipt of specific death stimuli. Such information would be useful in the design of strategies to regulate the activation of caspases during apoptosis.


RESULTS
We developed two novel fluorescent substrates that were specifically cleaved by caspase-1 or caspase-3. For in vitro studies, four-amino-acid recognition sequences, YVAD for caspase-1 and DEVD for caspase-3, were introduced between blue fluorescent protein (BFP) and green fluorescent protein (GFP), expressed in bacteria and purified. For in vivo studies, YVAD and DEVD were introduced between cyan fluorescent protein and yellow fluorescent protein, and expression was monitored in live mammalian cells. The proximity between fluorophores was determined using fluorescence resonance energy transfer. Purified substrates were cleaved following exposure to purified caspase-1 and caspase-3. In Cos-7 cells, caspase-1 and caspase-3 substrates were cleaved upon induction of apoptosis with staurosporine, a protein-kinase inhibitor, whereas caspase-3 but not caspase-1 substrate was cleaved upon treatment of cells with the DNA-damaging agent mitomycin c.


CONCLUSIONS
These substrates allow the spatial activation of specific members of the caspase family to be deciphered during the initiation and execution phase of programmed cell death, and allow activation of specific caspases to be monitored both in vivo and in vitro. This technology is also likely to be useful for high-throughput screening of reagents that modulate caspase activity.
Mahajan
N P
NP
The Lineberger Comprehensive Cancer Center, Department of Cell Biology and Anatomy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
Harrison-Shostak
D C
DC
Michaux
J
J
Herman
B
B
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D019556:COS Cells; D020170:Caspase 1 / Q000378:metabolism; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D010450:Endopeptidases / Q000378:metabolism*; D004789:Enzyme Activation; D005456:Fluorescent Dyes; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism*; D009154:Mutation; D013379:Substrate Specificity
D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D010450:Endopeptidases; D053148:Caspase 3; D020169:Caspases; D020170:Caspase 1