Abstract
Caspase-3 and -7 are considered functionally redundant proteases with similar proteolytic specificities. We performed a proteome-wide screen on a mouse macrophage lysate using the N-terminal combined fractional diagonal chromatography technology and identified 46 shared, three caspase-3-specific, and six caspase-7-specific cleavage sites. Further analysis of these cleavage sites and substitution mutation experiments revealed that for certain cleavage sites a lysine at the P5 position contributes to the discrimination between caspase-7 and -3 specificity. One of the caspase-7-specific substrates, the 40 S ribosomal protein S18, was studied in detail. The RPS18-derived P6-P5' undecapeptide retained complete specificity for caspase-7. The corresponding P6-P1 hexapeptide still displayed caspase-7 preference but lost strict specificity, suggesting that P' residues are additionally required for caspase-7-specific cleavage. Analysis of truncated peptide mutants revealed that in the case of RPS18 the P4-P1 residues constitute the core cleavage site but that P6, P5, P2', and P3' residues critically contribute to caspase-7 specificity. Interestingly, specific cleavage by caspase-7 relies on excluding recognition by caspase-3 and not on increasing binding for caspase-7.
Highlights
Caspase-3 and -7 are considered functionally redundant proteases with similar proteolytic specificities
Because caspases show hardly any specificity for tetrapeptides [23], these results indicate that additional residues in the P6 –P5Ј cleavage site region other than P4 –P1 may determine the specific recognition of RPS18 by caspase-7
The specificity of caspases for the P4 –P2 cleavage site positions has been extensively documented by peptidebased approaches such as combinatorial tetrapeptide screenings [8, 10, 11] and proteome-derived peptide libraries
Summary
Caspase-3 and -7 are considered functionally redundant proteases with similar proteolytic specificities. The specificity of caspases was rigorously profiled by using combinatorial tetrapeptide libraries [8], proteome-derived peptide libraries [9], and sets of individual peptide substrates [10, 11] The results of these studies indicate that specificity motifs for caspase-3 and -7 are nearly indistinguishable with the canonical peptide substrate, DEVD, used to monitor the enzymatic activity of both caspase-3 and -7 in biological samples. This overlap in cleavage specificity is manifested in their generation of similar cleavage fragments from a variety of apoptosis-related substrates such as inhibitor of caspaseactivated DNase, keratin 18, PARP, protein-disulfide isomerase, and Rho kinase I We identified 55 cleavage sites in 48 protein substrates, encompassing mutual, preferred, and unique caspase-3 and -7 cleavage sites
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