Apoptosome
The apoptosome is a quaternary protein structure formed in the process of apoptosis. It is formed by the release of cytochrome c from the mitochondrion responses to an internal or external cell death stimulus. Stimuli can differ from DNA damage or viral infection to developmental signals for instance like those leading to the degradation of a tadpole’s tail.
When cytochrome c is released, it binds to the cytosolic protein Apoptotic protease activating factor-1 to facilitate the formation of the apoptosome in mammalian cells. Biochemical and structural studies have shown that this interaction is essential for apoptosome assembly. Additionally, the nucleotide dATP binds to Apaf-1 as a third component, although its precise role in the process remains under investigation.
The mammalian apoptosome had never been crystallized, but a human Apaf-1/cytochrome-c apoptosome has been imaged at lower resolution by cryogenic transmission electron microscopy in 2002, showing a heptameric wheel-like particle with 7-fold symmetry. Recently, a medium resolution structure of human apoptosome was also solved by cryo-electron microscopy, which allows unambiguous inference for positions of all the Apaf-1 domains and cytochrome c. A crystal structure of the monomeric, inactive Apaf-1 subunit is currently obtainable. Following its formation, the apoptosome can then recruit and activate the inactive pro-caspase-9. Once activated, this initiator caspase can then activate effector caspases and trigger a cascade of events leading to apoptosis.
History
The term “apoptosome” was introduced firstly in Yoshihide Tsujimoto’s 1998 paper; “The role of Bcl-2 family proteins in apoptosis: Apoptosomes or mitochondria?” The apoptosome was previously recognized as a ternary complex involving caspase-9 and B-cell lymphoma-extra-large, which each bind to a specific APAF-1 domain. This complex was believed to play a regulatory role in mammalian cell death. An article published in The Journal of Chemistry, identified Apaf-1 as a regulator of apoptosis, responsible for activating procaspase-9.In 1999, the criteria defining an apoptosome were established. The first criteria were that it had to be large complex. Secondly, its formation requires the hydrolysis of a high energy bond of ATP and dATP. Finally, it must activate procaspase-9 in its functional form. The formation of this complex marks the point of no return in apoptosis. The stable multimeric complex of Apaf-1 and cytochrome c met these criteria and became known as the apoptosome.
The apoptosome was thought to be a multimeric complex for two reasons. The first reason being, to bring multiple procaspase-9 molecules close together for cleavage. The second reason being, to raise the threshold for apoptosis, therefore nonspecific leakage for cytochrome c, would not result in apoptosis.Once the apoptosome was identified as the activator of procaspase-9, research into mutations affecting this pathway gained significance and became an important research area. Studies explored its role in conditions such as human leukemia cells, ovarian cancer and viral infections. Research continues to investigate this pathway in further detail. There are hidden routes for cell death as well, which are independent of Apaf-1 and therefore the apoptosome. These routes are also independent of caspase-3 and 9. These hidden pathways for apoptosis are slower, but may prove useful with further research.
Structure
The apoptosome is a protein complex assembled around the adapter protein Apaf-1 during mitochondria-mediated apoptosis, which is stimulated by cellular stress.To form the apoptosome, ATP/dATP and cytochrome c must be present in the cytosol. In response to cellular stress, the mitochondria release cytochrome c into the cytoplasm. Cytochrome c then binds to the C-terminal region of Apaf-1, which contains WD-40 repeats. This interaction promotes the oligomerization of Apaf-1 molecules, forming a wheel-like apoptosome complex. During this process, procaspase-9 is recruited to the CARD domain located at the N-terminus of Apaf-1. Once assembled, the apoptosome activates caspases, which cause a controlled break down of the cell.The human apoptosome forms a heptameric, wheel-shaped complex with sevenfold rotational symmetry. Its three-dimensional structure was first determined at 27 Å resolution using electron cryomicroscopy, with a calculated mass of 1 megadalton . High-resolution cryogenic electron microscopy have revealed that each Apaf-1 subunit extends outward via HD2 arms into a V-shaped regulatory region composed of two β-propeller domains. These domains are formed by 15 WD40 repeats: one with seven blades and the other with eight. Cytochrome c binds within the cleft between the β-propellers, stabilizing the extended conformation of Apaf-1 and facilitating nucleotide exchange from ADP to ATP/dATP.
The central hub of the apoptosome is formed by the NOD, which includes the NBD, HD1 and WHD subdomains. These regions enable oligomerization and form the structural correlations of the complex. The CARD domains of Apaf-1 are flexibly attached above the central hub, and upon binding procaspase-9, they organize into a disk-like, acentric spiral structure on top of the hub.
The NOD domains of Apaf-1 form a structural platform lined with conserved helix-loop-helix motifs that create a central pore, helping stabilize the apoptosome. Its assembly depends on nucleotide exchange and requires structural changes in Apaf-1, triggered by cytochrome c binding.
Procaspase-9 binds to the apoptosome through its N-terminal CARD, which binds with Apaf-1 CARDs through specific binding surfaces. These CARDs form a left-handed spiral of Apaf-1/pc-9 CARD pairs. The most common configuration consists of four Apaf-1 and three or four procaspase-9 CARDs, forming a disk on top of the platform. This corresponds to approximately three to four procaspase-9 molecules recruited per seven Apaf-1 subunits. Not all Apaf-1 CARDs participate in the spiral due to linker length constraints.
Activation of procaspase-9 happens in two ways: by forming dimers with other procaspase-9 molecules and by pairing with Apaf-1 subunits. The apoptosome platform promotes proximity-induced dimerization of procaspase-9 molecules, enabling their activation. Catalytic domains of procaspase-9 may also form heterodimers with Apaf-1 subunits. These interactions may activate other proteins involved in cell death, such as caspase-3. Since the catalytic domains are connected to the CARD disk by flexible linkers, they can occupy variable positions on the central hub.
In mammalian cells, cytochrome c is essential for apoptosome assembly and helps stabilize the complex. However, in some invertebrates like C. elegans and Drosophila, the apoptosome can assemble and activate caspases without cytochrome c.
Several accessory proteins have been observed to co-purify with the apoptosome, including caspase-3, which may interact with the complex either directly or through active caspase-9. Caspase-3 can also cleave caspase-9, regulating its dissociation from the apoptosome and potentially amplifying the apoptotic signal.
Apaf-1 has an estimated molecular weight of about 140 kDa and consists of three major regions:
- The N-terminal CARD domain:
- The central NB-ARC/NOD domain:
- The C-terminal WD40 region:
Detailed structural features
- The nucleotide-binding domain contains conserved Walker A and Walker B motifs essential for nucleotide binding.
- HD1 and WHD interact to form the NOD core, mediating oligomerization and structural assembly.
- HD2 connects the NOD to the regulatory β-propeller region and contributes to the flexibility and positioning of the spokes.
- The two β-propellers form a cleft that binds cytochrome c. This interaction is stabilized by hydrogen bonds and salt bridges.
Non-human organisms
The above descriptions are for the human apoptosome. Apoptosome complex structures from other organisms have many similarities, but are of quite different sizes and numbers of subunits, as shown in the figure. The fruit-fly system, called Dark, has a ring of eight subunits. The nematode apoptosome, called CED-4, is octameric but much smaller, and it does not include the regions that would bind cytochrome C.Mechanism of action
Initiation
The initiation of apoptosome action aligns to the first steps in the programmed cell death pathway. Apoptosis can be triggered in one of two ways in animals. The first being: the extrinsic pathway, which involves the binding of extracellular ligands to transmembrane receptors. The second being the intrinsic pathway, which takes place in the mitochondria. This intrinsic pathway involves the release of cytochrome C from the mitochondria and subsequent binding to the cytosolic protein Apaf-1. The release of cytochrome c is therefore necessary for the initiation of apoptosome action, and this release is regulated in several ways, most notably by the detection of calcium ion levels.Cytochrome c release
release is thought to occur via two distinct mechanisms. The first involves the mitochondrial permeability transition pore, which opens in response to elevated mitochondrial Ca2+ levels and oxidative stress, leading to the release of intermembrane space proteins. The mPTP has several components, including the adenine nucleotide translocase, the voltage-dependent anion channel, and the mitochondrial F1Fo ATP synthase. The opening of the mPTP causes mitochondrial swelling, rupturing the outer mitochondrial membrane, which allows proteins like cytochrome c to leak into the cytosol. This permeability change is often associated with mitochondrial depolarization and the collapse of the mitochondrial membrane potential, resulting in a halt in ATP production. The discovery of the pharmaceutical agent cyclosporine A, which inhibits this process, has provided further insights into this mechanism.A second pathway, independent of the mPTP, involves the VDAC, which can be directly opened by pro-apoptotic members of the Bcl-2 protein family. These proteins induce the permeabilization of the outer mitochondrial membrane, facilitating the release of cytochrome c from the intermembrane space into the cytosol. This mechanism also contributes to the collapse of the mitochondrial membrane potential and a subsequent loss of mitochondrial function, promoting apoptotic or necrotic cell death.