FRET evidence that an isoform of caspase-7 binds but does not cleave its substrate.
A caspase-7 biosensor (vDEVDc) based on FRET (fluorescence resonance energy transfer) was used to study the proteolytic properties of caspase-7, an executioner protease in cellular apoptosis. An active isoform of caspase-7 with the 56 N-terminal residues truncated (57casp7) cleaved vDEVDc at the recognition sequence, resulting in a FRET efficiency decrease of 61%. In contrast, an isoform with the 23 N-terminal residues truncated (24casp7) bound to vDEVDc but did not cleave the substrate, resulting in a FRET increase of 15%. Kinetic results showed an exponential substrate cleavage and binding curve for the 57casp7 and 24casp7 isoforms, respectively. FRET changes of the vDEVDc biosensor were also monitored in cos-7 cells upon STS-induced apoptosis. Finally, we modeled caspase-7 binding to vDEVDc and estimated a FRET emission ratio increase of 31.7%, which agrees with the 15% experimental result. We showed that two differently truncated isoforms of caspase-7 exhibit different enzymatic properties, namely binding by 24casp7 and hydrolysis by 57casp7.
Institute of Biomaterials and Biomedical Engineering, University of Toronto, Rosebrugh Building, 164 College Street, Room 407, Toronto, Ont., Canada. isaac.li@utoronto.ca
Isaac T S
IT
Li
Elizabeth
E
Pham
Jason Jui-Hsuan
JJ
Chiang
Kevin
K
Truong
D000818:Animals; D015374:Biosensing Techniques; D019556:COS Cells; D053179:Caspase 7 / Q000032:analysis* / Q000378:metabolism; D002522:Chlorocebus aethiops; D003198:Computer Simulation; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D007527:Isoenzymes / Q000032:analysis / Q000378:metabolism; D007700:Kinetics; D013379:Substrate Specificity
D007527:Isoenzymes; D053179:Caspase 7