Proteome-wide substrate analysis indicates substrate exclusion as a mechanism to generate caspase-7 versus caspase-3 specificity.
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.
Demon
Dieter
D
Department for Molecular Biomedical Research, Flanders Institute for Biotechnology (VIB), Ghent 9052, Belgium.
Van Damme
Petra
P
Vanden Berghe
Tom
T
Deceuninck
Annelies
A
Van Durme
Joost
J
Verspurten
Jelle
J
Helsens
Kenny
K
Impens
Francis
F
Wejda
Magdalena
M
Schymkowitz
Joost
J
Rousseau
Frederic
F
Madder
Annemieke
A
Vandekerckhove
Joël
J
Declercq
Wim
W
Gevaert
Kris
K
Vandenabeele
Peter
P
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
D000596:Amino Acids; D010455:Peptides; D020543:Proteome; D053148:Caspase 3; D053179:Caspase 7