The Apoptosome Activates Caspase-9 by Dimerization Cristina Pop, John Timmer, Sabina Sperandio, Guy S. Salvesen  Molecular Cell  Volume 22, Issue 2, Pages 269-275 (April 2006) DOI: 10.1016/j.molcel.2006.03.009 Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 1 Proposed Activation Models and Constructs Used in The Study (A) The two contrasting caspase-9 activation models. The apoptosome is depicted by the seven Apaf-1 monomers that arrange in a ring to form this specific activation platform (Acehan et al., 2002). In the allosteric model, a single caspase-9 molecule is activated by rearrangements of the active site induced by interactions with apoptosome. In the induced proximity model the local concentration of caspase-9 overcomes a kinetic barrier to dimerization, and monomer/monomer contacts within the catalytic domain of the caspase-9 dimer cause the activating rearrangements. (B) The constructs used in the study. In caspase-9/8, the recruitment domains (DEDs) of caspase-8 are replaced by the CARD of caspase-9 to generate a hybrid protein capable of recruitment to the apoptosome. Mutation of the interchain Asp residues to Ala prevents processing to the two-chain form. Unless specifically stated, all experiments were performed with two-chain caspases-8, -9, or 9/8. Molecular Cell 2006 22, 269-275DOI: (10.1016/j.molcel.2006.03.009) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 2 Kinetics of Caspase-9 Activation (A) Concentration dependence of caspase-9 activity in 0.7 M Na-citrate (♦). The fit shows a n ∼ 1.5 demonstrating a mixed first and second-order mechanism for caspase activation in Na-citrate. Caspase-3 activity in assay buffer is the control (⋄). (B) Concentration dependence of caspase-9 activity in the presence of Apaf-1 (4 μM) (•). As a control for varying caspase-9 concentration, the total concentration of caspase-9 was kept constant by adding a C285A catalytic mutant (○). Activation depends on the second power of the enzyme concentration (n = 1.8). The control plot shows a similar pattern (n = 2.1) meaning that the nonlinear dependence of activity versus concentration is not due to Kd limitations between caspase-9 and Apaf-1 interaction during a variation in caspase concentration. White squares represent activity of caspase-9 in absence of Apaf-1. The error bars represent the standard deviation of five independent experiments. Molecular Cell 2006 22, 269-275DOI: (10.1016/j.molcel.2006.03.009) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 3 Cleavage of Procaspase-3 and Ac-LEHD-AFC by Differentially Activated Caspase-9 Procaspase-3(C285A) was incubated at 23°C with caspase-9 (15–500 nM), preactivated by either Apaf-1 (8 μM) or 1 M sodium Na-citrate. The arrows indicate the protein components, and the black arrow indicates the most readily visible cleavage product of procaspase-3. LS/SS: large/small subunit. The asterisk represents cleavage of Apaf-1 by caspase-9, which does not influence its activity (not shown). The estimated kcat/KM value for gel reactions was derived according to Equation 2. The catalytic parameters for Ac-LEHD-AFC cleavage by caspase-9 determined under the same conditions are shown on the right (kcat in s−1; KM in μM; kcat/KM in M−1 s−1). Molecular Cell 2006 22, 269-275DOI: (10.1016/j.molcel.2006.03.009) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 4 The Caspase-9/8 Hybrid Activates Executioner Caspases in an Apoptosome-Dependent Manner (A) DEVD-ase activity of lysates activated by the addition of cyt c and dATP, depleted of caspase-9 and reconstituted with recombinant caspases (∗ = one chain, or ∗∗ = two-chain caspases). The assay reads out the activity of executioner caspases activated by endogenous caspase-9 or reconstituted with the indicated caspases. The linear portion of the slope was used to compute the amount of activity of each of the apical caspases (see B). (B) Caspase-9/8 activated in lysates by cyt c and dATP can be inhibited by recombinant CrmA, suggesting that the DEVD-ase activity comes either directly from caspase-9/8 or from cleaved caspase-3. BIR-3 inhibits caspase-9 but does not inhibit caspase-9/8. The error bars represent the standard deviation of three independent experiments. (C) Caspase-9/8 is activated only in the presence of cyt c. Caspase-8 (catalytic domain only) is the negative control to demonstrate that the activation of caspase-9/8 is dependent on the CARD. The data represent the mean and standard deviation (error bars) of five independent experiments. The buffer controls represent: I, caspase-8∗; II, caspase-8∗∗; III, caspase-9/8∗; IV, caspase-9/8∗∗; V, caspase-9∗∗—all added to the hypotonic buffer in the absence of cell lysate. Molecular Cell 2006 22, 269-275DOI: (10.1016/j.molcel.2006.03.009) Copyright © 2006 Elsevier Inc. Terms and Conditions