Executioner caspase-3 and caspase-7 are functionally distinct proteases

Abstract

Members of the caspase family of cysteine proteases play central roles in coordinating the stereotypical events that occur during apoptosis. Because the major executioner caspases, caspase-3 and caspase-7, exhibit almost indistinguishable activity toward certain synthetic peptide substrates, this has led to the widespread view that these proteases occupy functionally redundant roles within the cell death machinery. However, the distinct phenotypes of mice deficient in either of these caspases, as well as mice deficient in both, is at odds with this view. These distinct phenotypes could be related to differences in the relative expression levels of caspase-3 and caspase-7 in vivo, or due to more fundamental differences between these proteases in terms of their ability to cleave natural substrates. Here we show that caspase-3 and caspase-7 exhibit differential activity toward multiple substrate proteins, including Bid, XIAP, gelsolin, caspase-6, and cochaperone p23. Caspase-3 was found to be generally more promiscuous than caspase-7 and appears to be the major executioner caspase during the demolition phase of apoptosis. Our observations provide a molecular basis for the different phenotypes seen in mice lacking either caspase and indicate that these proteases occupy nonredundant roles within the cell death machinery.

Fig. 1.

Hydrolysis of synthetic substrates by human caspase-3 and caspase-7. (A) Sequence alignment of human caspase-3 and caspase-7. Identical residues are highlighted in black. The conserved active site residues are boxed. (B) SDS/PAGE analysis of purified recombinant human caspase-3 and caspase-7 visualized by Coomassie staining. Five micrograms of bacterial-derived purified proteins were loaded in each lane. (C) Active-site titration of recombinant human caspase-3 and caspase-7 against DEVD-AFC. Caspases were incubated at 37°C for 30 min with the indicated amounts of the irreversible caspase inhibitor zVAD-fmk followed by measurement of residual caspase activity for each treatment by fluorimetry using Ac-DEVD-AFC as a substrate. (D) Linear rate of hydrolysis by equimolar amounts of active caspase-3 and caspase-7 was determined by fluorimetric assay using Ac-DEVD-AFC as a substrate. (E) Equimolar amounts (25 nM) of recombinant caspase-3 and caspase-7 were compared for their ability to hydrolyze the indicated synthetic peptide substrates. Results are representative of three independent experiments.

Fig. 2.

Proteolysis of natural substrates and caspases by recombinant caspase-3 and caspase-7. Jurkat cell-free extracts were incubated for 2 h at 37°C with the indicated concentrations of recombinant human caspase-3 and caspase-7 followed by immunoblotting for the indicated proteins. (A and B) Substrates that were cleaved with similar efficiency by both enzymes. (C and D) Substrates exhibiting differential proteolysis by caspase-3 and caspase-7. (E) Proteolysis of the indicated ³⁵S-labeled caspases, prepared by in vitro transcription/translation, by recombinant caspase-3 and caspase-7. Reactions were carried out for 2 h at 37°C followed by SDS/PAGE and fluorography analysis. (F and G) Proteolysis of endogenous caspases by recombinant caspase-3 and caspase-7. Jurkat cell-free extracts were incubated for 2 h at 37°C with the indicated concentrations of recombinant caspase-3 and caspase-7 followed by immunoblotting for the indicated caspases. Results are representative of three independent experiments.

Fig. 3.

Bid and cochaperone p23 exhibit differential proteolysis by human and mouse caspase-3 and caspase-7. (A and B) Recombinant Bid, Rho-GDI, and cochaperone p23 were coincubated for 2 h at 37°C with the indicated concentrations of caspase-3 and caspase-7, and reaction products were separated by SDS/PAGE and visualized by Coomassie staining (A) or immunoblotting (B). Note that recombinant Bid purified from bacteria as a doublet and both species were confirmed as Bid protein by mass spectrometry analysis. The reason for the difference in mobility of the two forms of Bid is not clear but may relate to partial misfolding of the protein. (C) Time-course analysis of purified Bid, Rho-GDI, and cochaperone p23 proteolysis by caspase-3 and caspase-7. Substrates were incubated at 37°C with 200 nM recombinant caspase-3 or caspase-7, and samples were taken at the indicated times. Reaction products were analyzed by SDS/PAGE and visualized by immunoblotting. (D) Densitometry analysis was performed to calculate the percentage of proteolysis over time for each of the treatments shown in Fig. S1 where 800 nM of each caspase was used to cleave recombinant purified Bid, Rho-GDI, or cochaperone p23. (E) Mouse J774 cell-free extracts were incubated for 2 h at 37°C with the indicated concentrations of recombinant mouse caspase-3 and caspase-7 followed by immunoblotting for the indicated proteins. Results are representative of three independent experiments.

Fig. 4.

Depletion of caspase-3 abrogates the majority of cytochrome c/dATP-induced caspase-dependent alterations to the proteome. (A) Mock-depleted or caspase-3-depleted Jurkat cell-free extracts were incubated for 2 h at 37°C in the presence or absence of cytochrome c/dATP as shown. Samples (350 μg per treatment) of each reaction were subjected to two-dimensional SDS/PAGE analysis, and proteins were subsequently visualized by silver staining. Protein spots corresponding to novel proteolytic fragments not detected on the untreated control gel are indicated by red arrows, and protein spots present in the control but not detected in cytochrome c/dATP-treated samples are indicated by yellow arrows. (B) Histogram indicating the approximate total number of alterations detected in cytochrome c/dATP-treated mock-depleted extracts or caspase-3-depleted extracts. (C) Zoomed area of the gels outlined in A where the protein indicated by an arrow was identified by mass spectrometry analysis as a cleaved fragment of cochaperone p23.

Fig. 5.

CASP-3-deficient MCF-7 cells fail to cleave multiple caspase substrates with the exception of cochaperone p23 and PARP. (A) Immunoblot analysis of the indicated caspase substrates within lysates generated from caspase-3-deficent (MCF7^vector) or caspase-3-reconstituted (MCF7^Casp-3) cells that were left untreated (UT) or were cultured for 22 h in the presence of actinomycin D (ActD; 5 μM) or cisplatin (Cis; 100 μM). (B) Cell death counts for corresponding treatments in A were performed based on morphological criteria as described in Materials and Methods. (C) Immunoblots of lysates generated from caspase-3-deficient (MCF7^vector) cells transfected with 1 μg of either CASP-3 or CASP-7 shRNA plasmids for 72 h followed by treatment with ActD or cisplatin for 15 h. (D) Corresponding cell death counts for the cells treated in C. Results are representative of three independent experiments.