title: FRET evidence that an isoform of caspase-7 binds but does not cleave its substrate.
abstract: Caspase-7 is one of the executioner proteases in cellular apoptosis. Its kinetics has been monitored using biosensors based on the principle of fluorescence resonance energy transfer (FRET). Here, a caspase-7 biosensor (named vDEVDc) using fluorescent proteins as the donor and acceptor of FRET was used to study the biochemical properties of caspase-7. An active isoform of caspase-7 with the 56 N-terminal residues truncated (named 57casp7) cleaved the vDEVDc biosensor at the recognition sequence, resulting in a FRET efficiency decrease of 61%. In contrast, another caspase-7 isoform with the 23 N-terminal residues truncated (named 24casp7) bound the vDEVDc biosensor without cleaving the substrate, resulting in a FRET increase of 15%. The kinetics of the two caspase-7 isoforms were studied by monitoring the FRET change of the vDEVDc biosensor over time, which showed an exponential substrate cleavage and binding curve for the 57casp7 and 24casp7 isoform, respectively. Lastly, we modeled caspase-7 binding to the vDEVDc biosensor and estimated a FRET emission ratio increase of 16.2% after binding to caspase-7, which agrees with the 15% experimental result. We showed that two isoforms of caspase-7 with differently truncated prodomain exhibit different enzymatic properties, namely binding by the 24casp7 isoform and hydrolysis by 57casp7. We also demonstrated that our FRET biosensor (vDEVDc) can be used to detect not only the substrate cleavage event, but also the substrate binding event.
authors.affiliation: Dept. of Electrical & Computer Engineering, University of Toronto, ON, Canada.
authors.forename: Isaac T
authors.initials: IT
authors.lastname: Li
authors.forename: Jason Jui-Hsuan
authors.initials: JJ
authors.lastname: Chiang
authors.forename: Kevin
authors.initials: K
authors.lastname: Truong
mesh_terms: D001665:Binding Sites; D015374:Biosensing Techniques / Q000379:methods*; D053179:Caspase 7 / Q000737:chemistry*; D004789:Enzyme Activation; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D007527:Isoenzymes / Q000737:chemistry*; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D013379:Substrate Specificity
chemical_list: D007527:Isoenzymes; D053179:Caspase 7