A Unified Model for Apical Caspase Activation Kelly M Boatright, Martin Renatus, Fiona L Scott, Sabina Sperandio, Hwain Shin, Irene M Pedersen, Jean-Ehrland Ricci, Wade A Edris, Daniel P Sutherlin, Douglas R Green, Guy S Salvesen  Molecular Cell  Volume 11, Issue 2, Pages 529-541 (February 2003) DOI: 10.1016/S1097-2765(03)00051-0

Figure 1 Endogenous Apical Caspases Are Monomers Cytosolic extracts of Jurkat cells were prepared and electrophoresed in (A) pore limit (Native) PAGE under nondenaturing conditions or (B) SDS-PAGE followed by electro-transfer and immunoblotting with specific antisera. The size of executioner caspases-3 and -7 is consistent with dimers of catalytic domains (2 × 36 kDa), whereas the size of initiator caspases-8, -9, and -10 is consistent with monomers of the basic catalytic domain (46–56 kDa). The monomeric conformation of procaspases-8 and -9, in comparison with dimeric procaspase-3, was confirmed by fractionating the extract on a Superdex 200 column (C). Asterisks represent nonspecific immunoreactive species. Molecular Cell 2003 11, 529-541DOI: (10.1016/S1097-2765(03)00051-0)

Figure 2 Identification of the Active Form of Caspase-8 (A) Gel filtration of recombinant ΔDED caspase-8 two chain on a Superdex 200 column reveals that roughly 40% of the protein (smooth curve; left ordinate, mAU = milli-absorbance units at 280 nm) eluted as a monomer (∼35 kDa), but the majority of activity against the substrate Ac-IETD-AFC (step curve; right ordinate, RFU = relative fluorescence units) is present in the fraction corresponding to dimers (∼53 kDa, ∼60% of the protein). (B) Gel filtration of ΔDED caspase-8 single chain on a Superdex 200 column reveals that almost all the protein (smooth curve; left ordinate, mAU = milli-absorbance units at 280 nm) eluted as a monomer (∼35 kDa), but again the majority of activity against the substrate Ac-IETD-AFC (step curve; right ordinate, RFU = relative fluorescence units) is present in the fraction corresponding to dimers (∼70 kDa). The insets demonstrate the integrity of the purified recombinant proteins run in SDS-PAGE before gel filtration. Molecular Cell 2003 11, 529-541DOI: (10.1016/S1097-2765(03)00051-0)

Figure 3 Effect of Kosmotropes on Caspase-8 and -9 Activity (A and B) Two chain ΔDED caspase-8 (A) and two chain ΔCARD caspase-9 (B) were incubated with the indicated salts to a final concentration of 1.0 M in standard assay buffer, followed 5 min later by the addition of substrate. Linear hydrolysis rates (RFU/min) were determined at 37°C. Fold increase in activity is expressed relative to the activity in NaCl. (C) The concentration dependence of caspase-8 and -9 activation by ammonium citrate showed that caspase-8 (•) requires a slightly higher concentration for maximal activation than caspase-9 (O). (D) Single chain zymogen forms (100 nM) of each of the caspases were incubated to a final concentration of 1.0 M ammonium citrate in standard assay buffer, followed 5 min later by the addition of substrate. Linear hydrolysis rates were determined at 37°C. Fold increase in activity is expressed relative to untreated procaspase. Significantly, the single chain zymogen forms of the executioner caspases-3 and -7 are not activated by kosmotropes. (E) Gel filtration fractions corresponding to the monomeric peak of single chain caspase-8 (ΔDED SC) and the two chain form (ΔDED TC) were assayed in the absence (gray bars) or presence (black bars) of 1.0 M ammonium citrate in standard assay buffer. Specific activities (RFU/min/μg) were normalized to the amount of protein in each fraction. (F) Similarly, the specific activities of the monomeric fractions of caspase-9 forms, full-length or ΔCARD, were determined in the absence (gray bars) and presence (black bars) of 1.0 M ammonium citrate in standard assay buffer. (E) and (F) use a log y axis to better describe the large increases in activity induced by the kosmotrope. Molecular Cell 2003 11, 529-541DOI: (10.1016/S1097-2765(03)00051-0)

Figure 4 Substrate Specificity of Caspases-8 and -9 in Kosmotrope (A) Two chain ΔCARD caspase-9 (E306A) was incubated in caspase buffer and added at a protein concentration of 625 nM, and assayed with the positional scanning substrate library. Hydrolysis rates were determined and presented as a fraction of the maximal rate in each subset (P2, P3, and P4). (B) Identical incubations were set up, except that the caspase-9 was first preincubated for 5 min in 1.0 M ammonium citrate in standard assay buffer. (C) Two chain ΔDED caspase-8 was incubated in caspase buffer and added at a protein concentration of 50 nM, and assayed with the positional scanning substrate library. Hydrolysis rates were determined and presented as a fraction of the maximal rate in each subset (P2, P3, and P4). (D) Identical incubations were set up, except that caspase-8 was first preincubated for 5 min in 1.0 M ammonium citrate in standard assay buffer. Molecular Cell 2003 11, 529-541DOI: (10.1016/S1097-2765(03)00051-0)

Figure 5 Cleavage Does Not Activate Caspase-8 Cytosolic extract from Jurkat cells was treated with amounts of granzyme B or caspase-6 sufficient to convert the full-length caspase-8 to the p45 and p10 derivatives by cleavage in the interchain linker. The treated cytosol was next incubated with Biotinyl-VAD-FMK for 30 min, and the biotinylated proteins were captured on agarose-conjugated streptavidin, eluted, and run in SDS-PAGE. (A) The gel was electrotransferred and probed with antiserum to caspase-8. The first lane corresponds to untreated extract. Each subsequent pair of lanes contains treated extract (input) and avidin-captured material. The final pair of lanes is a positive control in which recombinant two chain ΔDED caspase-8 was spiked into the extract at the same time as the affinity label to confirm that activated caspase-8 (p20) could be labeled by the affinity probe and captured. (B) The same samples were probed with antiserum to caspase-3 to demonstrate that the affinity probe labels the p19 and p17 derivatives of the large subunit of caspase-3 that has been activated by granzyme B and caspase-6. This panel serves as a positive control for the affinity capture method and also demonstrates that proteolysis is sufficient to activate procaspase-3. Asterisks represent nonspecific immunoreactive species. (C) A cytosolic extract was treated with the indicated concentrations of Biotinyl-VAD-FMK in 1.0 M in sodium citrate for 30 min at 37°C, followed by the addition of agarose-conjugated streptavidin to capture the affinity-bound proteins. The samples were electrophoresed in SDS-PAGE, electrotransferred, and immunoblotted with antiserum to caspase-8. The buffer control lane at right demonstrates that no affinity capture is obtained in the absence of the kosmotrope. Molecular Cell 2003 11, 529-541DOI: (10.1016/S1097-2765(03)00051-0)

Figure 6 Transfection of Caspase-8 and -9 Interface Mutants Wild-type, catalytic Cys285Ala mutants, or mutants at the dimer interface of caspases-8 and -9 (1 μg portions unless otherwise noted) were transfected into HEK293 cells to test the importance of the dimer interface in supporting activation. (A) Flow cytometry scans demonstrate that viability (propidium iodide/Annexin V negative population) of cells obtained by mechanical resuspension is around 61%. Viability is maintained following transfection of the catalytic mutant and decreased only slightly by transfection of the Thr390Asp mutant. In contrast, transfection with wild-type caspase-8 and the isosteric interface mutant Thr390Ser induces apoptosis. (B) The extent of caspase activation initiated by overexpression of the caspase-8 mutants was quantified by measuring general caspase activity on the substrate Ac-DEVD-AFC. (Inset) Equal amounts of total protein were loaded onto SDS-PAGE for quantitation of caspase-8 transfection. (C) Similarly, wild-type caspase-9 and the dimer interface Phe390Val isosteric replacement mutant induced comparable caspase activity when transfected, but the dimer interface Phe390Asp mutant was incapable of initiating caspase activation. (Inset) Equal amounts of protein were loaded onto SDS-PAGE for quantitation of caspase-9 transfection by immunoblotting for the C-terminal FLAG epitope. (D) HEK293 cells were cotransfected with 0.25 μg Fas plasmid and 1.75 μg of the indicated caspase constructs to determine whether the interface mutants were capable of sustaining an intrinsic pathway stimulus. (E) HEK293 cells were cotransfected with 0.25 μg Bax plasmid and 1.75 μg of the indicated caspase constructs to determine whether the interface mutants were capable of sustaining an intrinsic pathway stimulus. Molecular Cell 2003 11, 529-541DOI: (10.1016/S1097-2765(03)00051-0)

Figure 7 A Unified Model for Apical Caspase Activation We propose that apical caspases exist as monomers, demonstrating only a weak equilibrium with the dimeric form (Kd well above physiologic concentration). Recruitment to oligomeric activation complexes forces a local high concentration, converting a second order reaction to a first order one that allows the monomers to overcome the weak interaction equilibrium and adopt the dimeric conformation—hence, the origin of the first proteolytic signal. The apical caspases may only be active in the presence of an activator complex, since once this is removed the dimer should dissociate to inactive monomers. Molecular Cell 2003 11, 529-541DOI: (10.1016/S1097-2765(03)00051-0)