The Apoptosome: The Executioner of Mitochondria‐mediated Apoptosis

Abstract

The apoptosome is a molecular complex of two major components – the adapter protein apoptotic protease activating factor 1 (Apaf1) and the protease caspase‐9. These are assembled during apoptosis upon Apaf1 interaction with cytochrome c, which is released from the intermembrane space of mitochondria under precise cell death stimuli. Apoptosome assembly triggers effector caspase activation, which – in turn – drives cell demise. Several proteins bind and regulate apoptosome. This complex is found in vertebrates, even though similar functions are played by ortholog proteins through slightly different mechanisms also in lower eukaryotes. Given the involvement of apoptosome dysfunctions in human diseases, this complex is also a relevant molecular target in biomedicine.

Key concepts

  • It was a stunning discovery to find out that cytochrome c, essential for adenosine triphosphate (ATP) production and cell life, was involved in a cell death pathway.

  • Apoptosis regulation is essential for avoiding unwanted cell death and allowing the correct development and homoeostasis of the whole organism. Therefore there are several regulators of apoptosome activity.

  • Caspases and Apaf1 have a key phylogenetically conserved role in apoptosis.

  • Apoptosis is evolutionary well‐conserved in terms of its basic mechanisms; however, the role of mitochondria in apoptosis of organisms other than vertebrates needs to be clearly defined.

Figure 1.

The intrinsic (or mitochondria‐mediated) pathway of apoptosis and its correlation with the extrinsic pathway. The intrinsic pathway of apoptosis is highlighted in blue: cytochrome c release from mitochondria is modulated by proteins belonging to the Bcl‐2 family. In the cytosol, cytochrome c binds Apaf1 and induces apoptosome assembly and activation of caspase‐9 which, in turn, activates caspase‐3 and caspase‐7. Effector caspases are responsible for the executioner phase of apoptosis. The extrinsic pathway of apoptosis is highlighted in orange: the assembly of a complex called DISC (death‐inducing signalling complex) promotes activation of caspase‐8 which can cleave BID and trigger cytochrome c release from mitochondria and apoptosome formation or can directly cleave effector caspases.

Figure 2.

Structure of the main apoptosome components: Apaf1, caspase‐9 and cytochrome c. (a) Schematic representation of Apaf1 domain organization. Human Apaf1 can be divided into three functional regions. A CARD domain, a NB‐ARC region and a WD‐40 region. The NB‐ARC region is formed by a NOD region and a superhelical domain HD2. The NOD region contains the AAA+ ATPase region, in which the α/βNBD domain and an HD1 domain can be distinguished. The HD1 domain is followed by a WDH domain. The bridging helix between the CARD and the NBD domain is drawn in orange. The WD40 region is organized into two domains: The first one contains seven WD40 repeats, whereas the C‐terminal region contains six WD‐40 repeats. (b) Ribbon diagram of the structure of WD‐40‐deleted Apaf1 (1–591) bound to ADP. The CARD domain (green) packs against the α/β NBD domain (blue), the short helical domain HD1 (cyan) and the winged‐helix domainWHD (magenta). ADP binds the hinge region between the α/β domain and HD1 but is also coordinated by a few critical residues from the WHD domain which, by sensing the bound nucleotide, seems to be a key element for the regulation of the autoinhibited state of Apaf1. In this autoinhibited conformation, the CARD domain is not accessible for recruiting procaspase‐9; blocking the ATPase domain does not allow the oligomerization of Apaf1 (Protein Data Bank accession code 1Z6T) (excerpt from Riedl et al., ). (c) Model for the full‐length autoinhibited Apaf1 conformation. The same structure depicted in Figure b is here seen in another orientation; its probable association with the WD‐40 domain interacting with the N‐terminal region is visible (excerpt from Riedl et al., ). (d) Simplified scheme of Figure c (see also Figure ). (e) Schematic representation of caspase‐9 domain organization. Human procaspase‐9 contains a CARD domain, followed by a large subunit and a small subunit separated by linker regions. The catalytic cysteine residue is Cys287 (red mark). Two black marks indicate Asp315 (the interchain autocleavage between the large and the small subunit) and the caspase‐3 cleavage site Asp330, respectively. (f) Schematic representation of dimeric CARD‐deleted caspase‐9. Caspase‐9 is composed of two domains; the large subunit of each domain is coloured in grey and the small subunit is coloured in blue. The bound inhibitor molecule is depicted in grey. Only the catalytic side in the left domain is active and, therefore, able to bind the inhibitor. In the right domain, the same residues are transposed from their catalytic conformation into a novel structure incapable of catalysis (Protein Data Bank accession code 1JXQ) (excerpt from Renatus et al., ). (g) Human cytochrome c is a 105 aa long protein. (h) Schematic representation of holocytochrome. Lys 72 is enlighted, since it is essential for the stability of the interaction between cytochrome c and Apaf1. The heme group is colored in red (Protein Data Bank accession code 1AKK) (excerpt from Kalanxhi and Wallace, ).

Figure 3.

Shape and structure of the human apoptosome. (a) Top view of the 12.8 Å resolution 3D structure of the wheel‐shaped apoptosome. The central hub and the seven arms are revealed (excerpt from Yu et al., ). (b) Two models for domain arrangement in the NB‐ARC region. Top: In the bridge model the winged helix bridges two neighbouring ATPase domains (cyan and green) to form the oligomer. In this way an inner CARD (orange) ring appears to be surrounded by an outer ring formed by the ATPase and the WHD (red) domains. Bottom: In the AAA+‐like model the ATPase domains form by themselves the oligomer by binding back to back onto each other. In this model the CARD domains form a looser ring on the top of the NOD region (excerpt from Riedl and Salvesen, ). The colour code is not the same as in Figure .

Figure 4.

Apoptosome formation in C. elegans and in D. melanogaster. (a) In C. elegans, in nonapoptotic cells, dimers of CED‐4 asymmetrically interact with CED‐9 which is bound to mitochondria. Apoptotic induction promotes EGL‐1 upregulation. Egl‐1 binds CED‐9 and displaces it from CED‐4, which is thus free to oligomerize as a tetrameric complex in the presence of ATP. On oligomerization, CED‐4 activates CED‐3 which will perform the final steps of apoptosis. (b) In Drosophila, normally Dronc is inhibited by DIAP1 and apoptotic induction upregulates Dronc, Dark and Diap‐1 inhibitors (Diap‐1 inhib: Reaper, HID amd Grim). Diap‐1 inhibitors bind and induce DIAP1 degradation thus releasing Dronc inhibition which, in the presence of dATP, can bind oligomerized Dark. In the apoptosome Dronc is activated and, in turn, cleaves and activates the effector caspases Drice and Dcp1. Roles of mitochondria and Buffy and Debcl are unclear in this system.

Figure 5.

Phenotypes related to Apoptosome components’ gene targeting. Gross and histological analysis of the neural embryonic phenotype of caspase‐9 (a–d), Apaf1 knockout (e–h) and cytochrome c knockout/in (i–l) mice (excerpt from Kuida et al., ; Cecconi et al., and Hao et al., , respectively). Whole mounts of e16.5 caspase 9+/− and −/− embryos (a, b), e16.5 Apaf1 wt and −/− embryos (e, f) and KA/+ control and KA/KA mutant e14.5 embryos (i, j) often show brain overgrowth and protrusion (indicated by an asterisk). Transverse sections of e10.5 caspase‐9 mutant and corresponding wild‐type brains stained with toluidine‐blue (c, d). Transverse sections of e12.5 Apaf1 mutant and corresponding wild‐type brains (g, h). Coronal sections of e14.5 KA/KA mutant embryos and corresponding KA/+ brains (k, l). In most cases, overexpansion of the neuroephitelium causes ventricular occlusion. C, caudal; r, rostral.

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Further Reading

Chipuk JE and Green DR (2008) How do BCL‐2 proteins induce mitochondrial outer membrane permeabilization? Trends in Cell Biology 18: 157–164.

Fadeel B, Ottosson A and Pervaiz S (2008) Big wheel keeps on turning: apoptosome regulation and its role in chemoresistance. Cell Death and Differentiation 15: 443–452.

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Steller H (2008) Regulation of apoptosis in Drosophila. Cell Death and Differentiation 15: 1132–1138.

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Ferraro, Elisabetta, and Cecconi, Francesco(Sep 2009) The Apoptosome: The Executioner of Mitochondria‐mediated Apoptosis. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0021573]