Human inflammatory caspases (caspase‐1, ‐4, and ‐5) are key players in the innate immune response. These enzymes have been shown to cleave proinflammatory substrates, implicating them in many inflammatory disease states. Their activity is frequently assessed using in vitro fluorogenic assays, with all three human inflammatory caspases preferring the same WEHD tetrapeptide. The study examines the specificity of these enzymes C‐terminal to the cleaved aspartate residue with Förster resonance energy transfer peptide‐based assays using 7‐methoxycoumaryl alanine A(MCA) as the donor and lysine‐conjugated dabsyl K(Dab) as the quencher. The P 4 –P 1 peptide sequences A(MCA)EHD, A(MCA)VAD, and A(MCA)QPD are varied on the C‐terminal (prime) side of the peptide. Historically, caspase‐4 and caspase‐5 have been grouped together in their reactivity. Herein, caspase‐5 only appreciably cleaves the A(MCA)EHDGK(Dab) peptide, whereas caspase‐4 displays broader reactivity. All base sequences react more considerably with caspase‐4 when a glycine is included C‐terminal to Asp. The specificity of caspase‐1 at this position varies based on the P 3 –P 1 sequence of the peptide. These results highlight the interconnectedness of the prime and nonprime side amino acid sequences and the different behavior of each enzyme, which can be useful in understanding these potential drug targets.
Young et al. (Sun,) studied this question.