In its simplest representation, apoptosis is a two-step peptidase cascade in which initiator caspases (caspases-8, -9, and -10) activate executioner caspases (caspases-3, -6, and -7). Although many intricacies exist-such as the proteolysis of initiator caspases by executioner caspases, which further regulates their activity-apoptotic pathways ultimately converge on the activation of caspase-3, the most proteolytically proficient member of the family. This central role has led to the development of numerous enzymatic assays to detect caspase-3 activity, its activation, and the cleavage of hallmark substrates, such as poly(ADP-ribose) polymerase 1 (PARP1). Like other members of the caspase family, caspase-3 minimally recognizes a five-amino-acid motif, usually located in a well-exposed loop within its substrates. Caspase-3 cleavage-site motif preferences have been systematically studied using peptides but not proteins. Here, we use a simple recombinant protein-based double brilliance bioluminescence resonance energy transfer (BRET2) biosensor assay for caspase-3 that enables robust and quantitative kinetic measurements in vitro. We used the biosensor by assessing its ability to distinguish between optimal and suboptimal cleavage-site motifs using a panel of BRET2 biosensors incorporating all 20 amino acids at the critical P4 position (the fourth residue N-terminal to the scissile bond). Except for arginine and lysine, we successfully determined the catalytic specificity (kcat/KM) for all other residues at P4. Notably, the range of proteolytic efficacies observed with BRET biosensors was significantly narrower than that previously reported using peptide-based libraries. Finally, we confirmed the biosensor's utility in apoptotic cells, demonstrating its robustness and broad applicability.
Blais et al. (Tue,) studied this question.