Proteome-wide substrate analysis indicates substrate exclusion as a mechanism to generate caspase-7 versus caspase-3 specificity

Mol Cell Proteomics. 2009 Dec;8(12):2700-14. doi: 10.1074/mcp.M900310-MCP200. Epub 2009 Sep 16.


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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Amino Acids / metabolism
  • Animals
  • Caspase 3 / metabolism*
  • Caspase 7 / metabolism*
  • Humans
  • Mice
  • Models, Molecular
  • Molecular Sequence Data
  • Peptides / chemistry
  • Peptides / metabolism
  • Plasmids / genetics
  • Protein Processing, Post-Translational
  • Proteome / chemistry
  • Proteome / metabolism*
  • Reproducibility of Results
  • Structure-Activity Relationship
  • Substrate Specificity


  • Amino Acids
  • Peptides
  • Proteome
  • Caspase 3
  • Caspase 7