title: Proteome-wide substrate analysis indicates substrate exclusion as a mechanism to generate caspase-7 versus caspase-3 specificity.
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.
authors.affiliation: Department for Molecular Biomedical Research, Flanders Institute for Biotechnology (VIB), Ghent 9052, Belgium.
authors.forename: Dieter
authors.initials: D
authors.lastname: Demon
authors.forename: Petra
authors.initials: P
authors.lastname: Van Damme
authors.forename: Tom
authors.initials: T
authors.lastname: Vanden Berghe
authors.forename: Annelies
authors.initials: A
authors.lastname: Deceuninck
authors.forename: Joost
authors.initials: J
authors.lastname: Van Durme
authors.forename: Jelle
authors.initials: J
authors.lastname: Verspurten
authors.forename: Kenny
authors.initials: K
authors.lastname: Helsens
authors.forename: Francis
authors.initials: F
authors.lastname: Impens
authors.forename: Magdalena
authors.initials: M
authors.lastname: Wejda
authors.forename: Joost
authors.initials: J
authors.lastname: Schymkowitz
authors.forename: Frederic
authors.initials: F
authors.lastname: Rousseau
authors.forename: Annemieke
authors.initials: A
authors.lastname: Madder
authors.forename: Joël
authors.initials: J
authors.lastname: Vandekerckhove
authors.forename: Wim
authors.initials: W
authors.lastname: Declercq
authors.forename: Kris
authors.initials: K
authors.lastname: Gevaert
authors.forename: Peter
authors.initials: P
authors.lastname: Vandenabeele
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D053148:Caspase 3 / Q000378:metabolism*; D053179:Caspase 7 / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011499:Protein Processing, Post-Translational; D020543:Proteome / Q000737:chemistry / Q000378:metabolism*; D015203:Reproducibility of Results; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D000596:Amino Acids; D010455:Peptides; D020543:Proteome; D053148:Caspase 3; D053179:Caspase 7