The Two Pathways of Apoptosis
The apoptotic cascade can be induced through several different mechanisms. The two main mechanisms are the intrinsic and extrinsic pathway. The intrinsic pathway is activated if apoptosis-initiating events occur in the mitochondria, whereas the extrinsic pathway is triggered via the binding of signalling molecules at death receptors on the cell surface. While activation of apoptosis occurs through different mechanisms, they all result in the release of apoptotic effector proteases called caspases which carry out the apoptotic cascade. Ultimately, apoptotic cells are cleared by phagocytes (e.g. macrophages and dendritic cells) to avoid cell rupture and subsequent release of inflammatory products. This process is called programmed cell removal; and occurs independently of apoptosis (4). In this section an overviiew of the two main pathways of apoptosis is given.
1) The Intrinsic Pathway
Initiation of the intrinsic apoptosis pathway occurs following the release of cytochrome c, an apoptogenic factor, from the intermembrane space (IMS) of the mitochondria (1, 2, 7, 8). The events leading to cytochrome c release are complex and vary under different circumstances. Certain conditions resulting in cellular stress such as acidosis, low ATP concentration, and oxidative stress (decreased availablility of oxygen) can promote the opening of the mitochondrial permeability transition pore (MPTP) (2). The MPTP is located on the inner mitochondrial membrane (IMM) and allows transport of calcium ions. Upon opening of the MPTP, Ca2+ influx causes the mitochondria to swell and lyse, releasing cytochrome c into the cytoplasm (1, 2). An imbalance of Bcl-2 proteins, a family of apoptotic regulators, can also induce cytochrome c release. In this case, the balance of pro-apoptotic, anti-apoptotic, and BH3-only proteins, all members of the Bcl-2 family, affects the permeability of the outer mitochondrial membrane (2, 3, 7). BH3-only proteins are crucial to this balance since they are able to sequester anti-apoptotic proteins (2, 7, 8). In tumors, the anti-apoptotic protein Bcl-2 is often over-expressed which promotes tumor cell survival (4). This observation highlights the importance of a balance in pro- and anti-apoptotic effectors for tissue homeostasis and how their de-regulation promotes cancer progression.
Following its release, cytosolic cytochrome c initiates the formation of the apoptosome complex by recruiting Apaf-1, and facilitating its association with dATP. This interaction results in a conformational change in Apaf-1 that exposes its caspase-activation and recruitment domain (CARD), subsequently promoting interaction with procaspase-9, the precursor to caspase-9 (11). Caspases reside in an inactive precursor state or zymogen, of the respective caspases, in order to protect the cell from aberrant protease activity. Thus, procaspases must undergo an activation event in order to mediate apoptosis; the first caspase to be activated in the intrinsic pathway is caspase-9 and is accordingly designated the initiator caspase of this pathway (11). Following its interaction with the apoptosome, procaspase-9 is activated via dimerization, and is subsequently poised to activate downstream executioner or effector caspases. The effector caspases of the intrinsic pathway are caspases-3 and -7 (Boatright, 2003). Cleavage of zymogens by activated caspase-9 occurs in the linking domain of the effector pro-caspases, which induces conformational changes that mold the active site. After activation, the procaspases participate in multiple maturation events (1). These events include auto-proteolytic cleavage of the linking domain and the pro-region of the procaspase, leaving the mature caspase. It is important to distinguish cleavage of the pro-region from effector caspase activation.
In the intrinsic pathway, the caspase cascade can be split into two phases. The first part is the initiation phase, which involves activation by dimerization of the procaspases. The second is the execution phase, with activation of caspases by proteolytic processing of dimerized zymogens.
2) The Extrinsic pathway
The death-receptor mediated (extrinsic) apoptotic pathway is very similar to the intrinsic pathway. The only major difference is that apoptotic signalling is initiated through membrane-bound death receptors such as Fas and tumour necrosis factor (TNF) receptor 1 (1). In cancer cells, these death receptors can be downregulated on the cell surface, desensitizing cancer cells to stimuli for extrinsic apoptosis (5). Much like the apoptosome, the death-inducing signaling complex (DISC) activates caspases of the extrinsic pathway, namely caspase-8 and caspase-10 (1,4). DISC components are oligomers of Fas and Fas-associated death domain (FADD) proteins (1, 4). Adaptors are recruited from DISC to bind domains on the procaspases and increase local caspase concentration. These domains, the extrinsic equivalents of the CARDS in the intrinsic pathway, are called death effector domains (DEDs) (1).
Active initiator caspases-8 and -10 can cleave executioner procaspases-7,-6, and -3, which converges the extrinsic and intrinsic caspase cascades. The release of cytochrome c from the mitochondria can also be an exacerbating stimulus for the caspase cascade of the extrinsic pathway (1, 2).
Mature caspase-9, -3, and -7 are regulated by inhibitors of apoptosis (IAPs), namely X-linked inhibitor of apoptosis (XIAP) (3, 4). These inhibitors have a domain that accommodates ubiquitination, which signals for proteasome degradation of the caspase. Increased levels of IAP family inhibitors, which inhibit apoptosis and promote cell survival, have been documented in pancreatic cancer and melanoma, (4).
Substrate Cleavage Ultimately Kills the Cell
Substrates for the effector caspases include proteins required for maintenance of cell membranes, organelle function, and enzymes which degrade the cell (such as nucleases). Other substrates are proteins that do not change the morphology of the cell but are necessary for metabolism and biogenesis (1, 3)The effector caspases in both the extrinsic pathway and intrinsic pathway activate downstream caspases via a cascade. The downstream effector caspases cleave cellular components until the integrity of the cell is compromised.
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