PMC

Procaspase-9 autoprocessing by the Apaf-1 apoptosome. (A) A schematic of procaspase-9 is shown with its CARD, large subunit (LS), activation loop, and small subunit (SS). Crucial amino acids in the active site (C287), the CARD (R56), and the activation loop (E306, D315, and D330) are highlighted. Purified wild-type and mutant Apaf-1, caspase-9, and caspase-3 proteins (10 μg each) were subjected to SDS–PAGE and stained with Coomassie blue. (B) Recombinant Apaf-1 and ProC9 were incubated with and without Cc/dATP. The samples were subsequently fractionated by Superose-6 gel-filtration chromatography, resolved using SDS–PAGE, and immunoblotted using Apaf-1 and caspase-9 antibodies (). (C) CARD-displacement and CARD-static models for the autoprocessing of ProC9. Details are described in the text.

Procaspase-9 undergoes rapid autoprocessing so that processed caspase-9 is primarily responsible for the activation of procaspase-3. (A) Apoptosome complexes were reconstituted with either non-cleavable ProC9-TM or wild-type ProC9 (12.5 nM) and incubated for 5–30 min, after which ProC3 (500 nM) was added to each incubation and immediately assayed for caspase-3 DEVDase activity. (B, C) ProC9-TM, ProC9, and C9-p35/p12 were incubated with Apaf-1 and Cc/dATP with and without GST–BIR3-RING of XIAP. A progression curve from one experiment is shown. Each bar represents the mean inhibition of caspase-9 activity of three independent experiments±s.e.m., in which the percent inhibition was calculated as follows: 100−[(V₀BIR3-RING/V₀) × 100]. Inset, purified wild-type ProC9 and GST–XIAP (BIR3-RING) were subjected to SDS–PAGE and stained with Coomassie blue. (D) Model of the Apaf-1 apoptosome as a molecular timer. Details are described in the text.

Autoprocessing of procaspase-9 reduces its affinity for the apoptosome. (A) Apoptosome complexes were assembled with decreasing concentrations of caspase-9 proteins (C9-p35/p12 and ProC9-TM; 200–0 nM), and the activation of ProC3 was determined by measuring DEVDase activity. Recombinant Apaf-1 and ProC3 were present in each incubation at 300 and 500 nM, respectively. Each bar represents the mean of four separate experiments±s.e.m. (B) K_M values for apoptosome-bound C9-p35/p12 (25 nM) and ProC9-TM (25 nM) were determined, as described in the Methods section. The graph shown is from a single experiment; K_M values in the inset represent the mean of three independent experiments±s.e.m., using at least two different protein preparations of caspase-9 and Apaf-1. (C) ProC9-TM or C9-p35/p12 were incubated with Apaf-1 and Cc/dATP, titrated with increasing concentrations of ProC9^*, and finally incubated with ProC3. Apoptosome-dependent activation of ProC3 was then determined by measuring caspase-3 DEVDase activity as already described. The graph shown is from a single experiment; IC₅₀ values in the inset represent the mean of three independent experiments±s.e.m., using at least two different protein preparations of caspase-9 and Apaf-1.

Prolonging or disengaging the proteolytic-based timer profoundly sensitizes cells to apoptosis. (A) Caspase-9-deficient lysates were reconstituted with increasing concentrations of wild-type ProC9 (0–100 nM) or non-cleavable ProC9-TM (25 nM). After the addition of Cc/dATP, apoptosome-mediated activation of endogenous ProC3 was determined by measuring caspase-3 DEVDase activity, as described in the Methods section. The results are typical of three independent experiments. (B) Caspase-9 deficient MEFs were infected with lentiviruses expressing empty vector, wild-type ProC9, or non-cleavable ProC9-TM. Stable clones, expressing varying concentrations of ProC9 or ProC9-TM (L=low; M=medium; H=high), were obtained following selection with hygromycin, and the cells were subsequently UV irradiated (8 min) on a UV illuminator and assayed for cell death by Annexin V/propidium iodide staining and flow cytometry. Each bar represents the mean of six independent experiments±s.e.m.

Procaspase-9 is autoprocessed through a CARD-displacement mechanism. (A) T7-tagged C9-p35/p12 and ProC9-TM were incubated with T7-tagged ProC3^* in the presence of Apaf-1 and Cc/dATP. Reactions were carried out, as described in the experimental procedures, resolved by SDS–PAGE, and immunoblotted using an anti-T7 antibody. As C9-p35/p12 and ProC3^* co-migrated (top panel), the membranes in these experiments were also stripped and re-blotted for caspase-9 (anti-C9) and caspase-3 (anti-C3) using caspase-specific antibodies. (B) The initial velocities (V₀) of apoptosome-bound C9-p35/p12, ProC9-TM, and C9-p35/p12-R56A on the substrate ProC3 were assessed by measuring caspase-3 DEVDase activity, as described in the Methods section. Each bar represents the mean of three separate experiments±s.e.m. (C) Recombinant wild-type C9-p35/p12 or C9-p35/p12-R56A were incubated with Apaf-1 and Cc/dATP. The samples were subsequently fractionated using Superose-6 gel-filtration chromatography, resolved by SDS–PAGE, and immunoblotted using Apaf-1 and caspase-9-specific antibodies (). (D) T7-tagged C9-p35/p12 and ProC9-TM were incubated with Flag-tagged ProC9^*-R56A in the presence of Apaf-1 and Cc/dATP. Reactions were carried out, as described in the Methods section, and immunoblotted for the processing of Flag–ProC9^*-R56A using an anti-Flag antibody. In lanes 7 and 14, active caspase-3 (1 μM) was added as a positive control to confirm that ProC9^*-R56A could be cleaved within its activation loop. (E, F) Apoptosome-bound T7-tagged C9-p35/p12 or ProC9-TM were incubated with T7-tagged ProC9^*-R56A and either T7-tagged ProC3^* or ProC3. Samples were then analysed by SDS–PAGE and immunoblotted using an anti-T7 antibody or assayed for active caspase-3 DEVDase activity. The membranes in panel (E) were also stripped and re-blotted for caspase-9 (using anti-C9) and caspase-3 (using anti-C3) using caspase-specific antibodies. Each bar represents the mean of three separate experiments±s.e.m.