and Peter Vandenabeele a,b,oCaspase-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.

Background:To study the potential role of caspase-2, we determined its cleavage site specificity and compared it with that of caspase-3 and -7. Results: N-terminal COFRADIC analysis of native proteomes revealed that caspase-2, -3, and -7 have overlapping specificities. Conclusion: Caspase-2, -3, and -7 share nearly indistinguishable cleavage site specificity. Significance: This finding suggests that caspase-2 has a proapoptotic function.

We investigated cytotoxic activity of antimicrobial peptides of different origin (both naturally occurring and synthetic), structure and known mechanisms of action against human histiocytic lymphoma cell line U937. The strongest cytotoxic activity against U937 cell line was shown by Pexiganan MSI-78, followed by Citropin 1.1, Protegrin 1 and a synthetic lipopeptide, N-α-palmitoyl-L-lysyl-L-lysine amide (Pal-Lys-Lys-NH₂). The cytotoxic activity of the peptides was more dependent on the time of incubation than concentration. Only for the lipopeptide, whose mode of action was restricted to disruption of electric potential of the cell membrane, the correlation between cytotoxicity and concentration was almost linear. The high cytotoxicity of Pexiganan MSI-78, Protegrin 1 and the lipopeptide could be basically explained by their membranolytic activity leading to necrosis. However, in the case of Citropin 1.1, the cell membrane integrity was disrupted only slightly and independently of the peptide concentration. Therefore, some other mechanism of action might be responsible for its strong dose-dependent cytotoxic activity, e.g., membranolytic activity leading to apoptosis. Furthermore, TNF-α production due to LPS (lipopolysaccharide) stimulation was suppressed by the presence of Citropin 1.1, Pexiganan MSI-78 or Protegrin 1, but not by Buforin 2 or the lipopeptide. Our experiments have shown that cytotoxic activity is not limited to some specific molecular structure of a peptide, but rather to the length of the peptide chain as it is likely to affect the efficiency of the tumor cell membrane disruption and interaction with LPS.