Apoptosome

The apoptosome is a large quaternary protein structure formed in the process of apoptosis. Its formation is triggered by the release of cytochrome c from the mitochondria in response to an internal (intrinsic) or external (extrinsic) cell death stimulus. Stimuli can vary from DNA damage and viral infection to developmental cues such as those leading to the degradation of a tadpole's tail. The apoptosome is a large quaternary protein structure formed in the process of apoptosis. Its formation is triggered by the release of cytochrome c from the mitochondria in response to an internal (intrinsic) or external (extrinsic) cell death stimulus. Stimuli can vary from DNA damage and viral infection to developmental cues such as those leading to the degradation of a tadpole's tail. In mammalian cells, once cytochrome c is released, it binds to the cytosolic protein Apaf-1 to facilitate the formation of an apoptosome. An early biochemical study suggests a two-to-one ratio of cytochrome c to apaf-1 for apoptosome formation. However, recent structural studies suggest the cytochrome c to apaf-1 ratio is one-to-one. It has also been shown that the nucleotide dATP as third component binds to apaf-1, however its exact role is still debated. The mammalian apoptosome had never been crystallized, but a human APAF-1/cytochrome-c apoptosome has been imaged at lower (2 nm) resolution by cryogenic transmission electron microscopy in 2002, revealing a heptameric wheel-like particle with 7-fold symmetry. Recently, a medium resolution (9.5 Ångström) structure of human apoptosome was also solved by cryo-electron microscopy, which allows unambiguous inference for positions of all the APAF-1 domains (CARD, NBARC and WD40) and cytochrome c. There is also now a crystal structure of the monomeric, inactive Apaf-1 subunit (PDB 3SFZ). Once formed, 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. The term Apoptosome was first introduced in Yoshihide Tsujimoto's 1998 paper 'Role of Bcl-2 family proteins in apoptosis: apoptosomes or mitochondria?'. However, the Apoptosome was known before this time as a ternary complex. This complex involved caspase-9 and Bcl-XL which each bound a specific Apaf-1 domain. The formation of this complex was then believed to play a regulatory role in mammalian cell death. In December of the same year, a further article was released in The Journal of Biological Chemistry stating that Apaf-1 is the regulator of apoptosis, through activation of procaspase-9. The criteria for an apoptosome were laid out in 1999. Firstly, it must be a large complex (greater than 1.3 million Daltons). Secondly its formation requires the hydrolysis of a high energy bond of ATP or dATP. And lastly it must activate procaspase-9 in its functional form. The formation of this complex is the point of no return, and apoptosis will occur. The stable APAF-1 and cytochrome mutimeric complex fit this description, and is now called the apoptosome. The apoptosome was thought to be a mutimeric complex for two reasons. Firstly, to bring multiple procaspase-9 molecules close together for cleavage. And secondly, to raise the threshold for apoptosis, therefore nonspecific leakage of cytochrome c would not result in apoptosis. Once the apoptosome was established as the procaspase-9 activator, mutations within this pathway became an important research area. Some examples include human leukemia cells, ovarian cancer and viral infections. Current research areas for this pathway will be discussed 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. The apoptosome is a multimolecular holoenzyme complex assembled around the adaptor protein Apaf1 (apoptotic protease activating factor 1) upon mitochondria-mediated apoptosis which must be stimulated by some type of stress signal T formation of the apoptosome requires the presence of ATP/dATP and cytochrome c in the cytosol.A stress stimulus can trigger the release of cytochrome c into the cytoplasm which will then bind to the C-terminus of Apaf-1 within a region containing multiple WD-40 repeats. The oligomerization of Apaf-1 appears to be accompanied by synchronized recruitment of procaspase-9 to the CARD motif at the Apaf-1 N-terminus. The apoptosome triggers the activation of caspases in the intrinsic pathway of apoptosis. The wheel-shaped heptameric complex with sevenfold symmetry structure of the apoptosome was first revealed at 27 Å resolution by electron cryomicroscopy techniques and has a calculated mass of about 1 MDa (Acehan et al. 2002). This wheel-like particle has seven spokes and a central hub. The distal region of the spoke has a pronounced Y shape. The hub domain is connected to the Y domain by a bent arm. Each Y domain comprises two lobes (a large one and a small one) between which cytochrome c binding sites. Because the resolution of the apoptosome structure was relatively low, two controversial models for apoptosome assembly were proposed. One model suggests NOD domains form the central hub and the CARD domains form a freer ring at the top of the NOD region. Another model proposes that Apaf-1 is organized in an extended fashion such that both the N-terminal CARD and the nucleotide binding region form the central hub of the apoptosome, whereas the 13 WD-40 repeats constitute the two lobes. The large lobe is formed by seven repeats and the small lobe is formed by six repeats. Each caspase- 9 molecule binds a CARD domain at the central hub, forming a dome shaped structure. This controversy has been resolved by a recent high resolution structure of the human apoptosome-procaspase-9 CARD complex. This structure clearly demonstrated that only the NOD regions form the central hub of the apoptosome (see pictures), while CARD is flexibly linked to the platform of apoptosome and becomes disordered in the ground state apoptosome. Once apoptosome binds to procaspase-9, the Apaf-1 CARDs and procaspase-9 CARDs form a flexible disk-like structure sitting above the platform. The number of WD-40 repeats has also been proved to be 15 instead of 13, and it is composed of a 7-bladed beta-propeller and an 8-bladed beta-propeller.