Internally quenched fluorescent peptide substrates disclose the subsite preferences of human caspases 1, 3, 6, 7 and 8

Subsite interactions are considered to define the stringent specificity of proteases for their natural substrates. To probe this issue in the proteoly tic pathways leading to apoptosis we have examined the P-4, P-1 and P-1' su bsite preferences of human caspases 1, 3, 6, 7 and 8, using internally quen ched fluorescent peptide substrates containing o-aminobenzoyl (also known a s anthranilic acid) and 3-nitro-tyrosine. Previous work has demonstrated th e importance of the S-4 subsite in directing specificity within the caspase family. Here we demonstrate the influence of the S-1 and S-1' subsites tha t flank the scissile peptide bond. The S-1 subsite, the major specificity-d etermining site of the caspases, demonstrates tremendous selectivity, with a 20000-fold preference for cleaving substrates containing aspartic acid ov er glutamic acid at this position. Thus caspases are among the most selecti ve of known endopeptidases. We find that the caspases show an unexpected de gree of discrimination in the P-1' position, with a general preference for small amino acid residues such as alanine, glycine and serine, with glycine being the preferred substituent. Large aromatic residues are also surprisi ngly well-tolerated, but charged residues are prohibited. While this descri bes the general order of P-1' subsite preferences within the caspase family , there are some differences in individual profiles, with caspase-3 being p articularly promiscuous. Overall, the subsite preferences can be used to pr edict natural substrates, but in certain cases the cleavage site within a p resumed natural substrate cannot be predicted by looking for the preferred peptide cleavage sites. In the latter case we conclude that second-site int eractions may overcome otherwise sub-optimal cleavage sequences.