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.
Li
Isaac T S
IT
Institute of Biomaterials and Biomedical Engineering, University of Toronto, Rosebrugh Building, 164 College Street, Room 407, Toronto, Ont., Canada. isaac.li@utoronto.ca
Pham
Elizabeth
E
Chiang
Jason Jui-Hsuan
JJ
Truong
Kevin
K
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