Proteome-wide Substrate Analysis Indicates Substrate Exclusion as a Mechanism to Generate Caspase-7 Versus Caspase-3 Specificity

Abstract

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.

Degradomics Reveals That Cleavage Specificity Profiles of Caspase-2 and Effector Caspases Are Alike

“…Although caspase-3 in loop 3 contains a Ser 209 residue that binds P5 Leu of the LDESD sequence, caspase-7 contains Pro 235 , which does not allow such interaction. Additional amino acids constituting the S5 pocket residing in loop 4 are hydrophilic in caspase-7 (Gln-276, Ser-277, and Asp-278), whereas in caspase-3 two of the three are hydrophobic (Phe-250, Ser-251, and Leu-252) (42). Contrary to these structural data, we observed no marked difference between caspase-3 and -7 in the amino acid sequence recognized in substrates derived from whole cell proteomes.…”

“…To distinguish between control and caspase-2-treated samples, differential acetylation was introduced; N-hydroxysuccinimide trideutero-acetate was used for all caspase-treated samples, whereas N-hydroxysuccinimide acetate was used for the control sample. Lysates were further treated with 5 mM iodoacetamide to block endogenous cysteine protease activity (51) before incubation with recombinant human caspases at a concentration of 200 nM for 1 h at 37°C (42). Proteases were then inactivated, and equal amounts of samples from the four setups were mixed and analyzed by N-terminal COFRADIC, including strong cation exchange chromatography and N-terminal peptide sorting steps (40,41,45) (Fig.…”

“…F-12K medium was supplemented with 0.3 mM L-arginine (15% of the standard concentration, at which the conversion of L-arginine to proline was not observed) using [ 12 After SILAC labeling, A549 cells were harvested and lysed as described (42). After blocking the activity of endogenous caspases by iodoacetamide (42) (39,43).…”

“…F-12K medium was supplemented with 0.3 mM L-arginine (15% of the standard concentration, at which the conversion of L-arginine to proline was not observed) using [ 12 After SILAC labeling, A549 cells were harvested and lysed as described (42). After blocking the activity of endogenous caspases by iodoacetamide (42) (39,43). As part of the N-terminal COFRADIC protocol, differential acetylation was then used to distinguish further between the two light-labeled samples (N-hydroxysuccinimide trideutero-acetate was used for the caspase-2, -3, and -7 samples and N-hydroxysuccinimide acetate for the control sample) and to distinguish between in vitro and in vivo N-acetylation of the caspase-treated samples, the former indicative for proteolytic events (43).…”

Conservation of the Extended Substrate Specificity Profiles Among Homologous Granzymes Across Species

“…Following 90 min incubation at 30°C, translates were incubated with iodoacetamide (f.c. 5 mM) for 20 min at 37°C as to avoid downstream caspase activity upon granzyme treatment (34). The translates (5 l) were subsequently incubated for 1.5 h at 37°C in granzyme assay buffer with the indicated concentrations of recombinant granzymes in a total volume of 30 l. Cleavage reactions were stopped by the addition of NuPAGE ® LDS Sample Buffer (Invitrogen) and heating the samples for 10 min at 70°C.…”