Caspases apoptosome

BH3 domain) of the BH3-only proteins binds to other Bcl-2 family members thereby influencing their conformation. This interaction facilitates the release of cytochrome C and other mitochondrial proteins from the intermembrane space of mitochondria. Despite much effort the exact biochemical mechanism which governs this release is not yet fully understood. The release of cytochrome C facilitates the formation of the apoptosome, the second platform for apoptosis initiation besides the DISC. At the apoptosome which is also a multi-protein complex the initiator caspase-9 is activated. At this point the two pathways converge. 

There is also crosstalk between the two pathways above the mitochondria. The BH3-only protein BID is cleaved by caspase-8 and -10 which yields truncated BID (tBED), the active pro-apoptotic fragment of BID. Thereby, even in cells in which the direct apoptosis pathway which result from death receptor crosslinking is blocked, e.g. by high expression levels ofthex-linked IAP (XIAP), the activity of tBED on mitochondria can result in the activation of caspase-3 because the IAP-imposed block on full caspase-3 activation and caspase-9 activity at the apoptosome is released by Smac/ DIABLO. 

Intrinsic system Apoptosis is initiated due to changes to the mitochondria during which the inner membrane becomes permeable to large molecules, probably as a result of a decrease in the membrane potential. This can result from intracellular damage (e.g. accumulation of Ca ions), lack of oxygen or fuel. This results in release of cytochrome c and other proteins from the mitochondria which stimulate apoptosis. In fact, these apoptotic proteins plus cytochrome c form a complex, the apoptosome, which activates an initiator caspase. 

In aminoglycoside-treated animals, the cells can be led to canonical apop-totic death through activation of caspases. Caspase-9 forms an apoptosome complex with cytochrome c and APAF-1 and leads to apoptosis through activation of caspase-3. Aminoglycosides activate caspases in auditory structures conversely, inhibition of caspase activity successfully blocks neomycin-induced vestibulotoxicity. In contrast, apoptotic markers were essentially absent in a mouse model of chronic kanamycin ototoxicity where death of auditory sensory cells ensued via cathepsins. The activation of cathepsin D was accompanied by the nuclear translocation of endonuclease G, necrotic cleavage of PARP, and activation of p,-calpain, all facets of necrotic cell death. 

Figure 14-8. Overview of pathways that regulate programmed cell death. Apoptosis may occur in response to signaling through either the extrinsic pathway or the intrinsic pathway. In each case, proteolytic cleavage activates an initiator caspase, caspase 8 or 9, either of which can cleave an effector caspase such as caspase 3. Apaf-1 is part of a large complex called the apoptosome that mediates the intrinsic pathway. Binding of an extracellular death ligand to its cell-surface receptor activates the extrinsic pathway.

Tlie initiator caspases receive proapoptotic signals and initiate the activation of a caspase cascade. Tliey are activated by an interaction with a transmembrane receptor or by cytotoxic influences. A complex is thus formed known as the apoptosome (see 15.4). The effector caspases are activated by an upstream caspase via a cascade mechanism. They are the component that executes apoptosis, initiates degradation of central proteins and directs the cell to death. 

A further cofactor in activation in this system is cytochrome c. The ATP-dependent activation occins in a complex of caspase, Apafl protein and Cyt c, which is also known as the apoptosome. 

Bcl-2 and related cytoplasmic proteins are key regulators of apoptosis [26], Anti-apoptotic proteins such as Bcl-2 and Bc1-Xl prevent apoptosis in response to numerous stimuli. During the apoptotic process, cytochrome c is released from mitochondria, but the release can be inhibited by the presence of Bcl-2 on the organelles [27]. The released cytochrome c forms an essential part of die apoptosome, which is composed of cytochrome c, Apaf-1, and procaspase-9 [28]. The complex formation results in activation of caspase-9, which leads to the stimulation of caspase-3. Bcl-XL has recently been reported to bind to Apaf-1 [29], It may inhibit the association of Apaf-1 with procaspase-9 and thereby prevent caspase activation. 

Apoptosome Cytosolic complex Apafl caspase-9 cytochrome c... 

Cytochrome c (Cyt c), the peripheral protein loosely associated with the inner membrane of mitochondria, is one of the most well-known factors involved in apoptosis (Green 2005). In healthy cells, Cyt c functions as an electron shuttle in the respiratory chain and its activity is necessary for life. Cyt c is released by the mitochondria as the consequence of elevated permeability of the outer membrane in responses to proapoptotic stimuli (Li et al. 1997). In the cytosol, Cyt c binds to the apoptosis-protease activating factor 1 (Apaf-1), which then recruits caspase-9 to form the apoptosome (Li et al. 1997). Caspase-9 in turn cleaves and activates executioner caspase-3, resulting in apoptotic cell death as described above. The whole process requires energy and relatively intact cell machinery. 

Following the outer mitochondrial membrane permeabilization, the apoptogenic factors are released into the cytoplasm. Among them, cytochrome c has an important role in caspase activation, because it is the cofactor for assembling a large caspase 9 activating complex in the cytoplasm, called apoptosome. Along with cytochrome c, the Apaf-1 protein and dATP or ATP are required to form this complex in the cytoplasm (Hill et al., 2003). 

The DISC-induced procaspase assembly results in the autoactivation of caspases 8 and 10. The DISC process of caspase activation seems to be analogous to the apoptosome process of caspase 9 activation. Caspase 8, in turn, cleaves and activates caspase 3, which is responsible for the apoptotic signal amplification with subsequent cell collapse. 

Bratton SB, Walker G, Srinivasula SM, Sun XM, Butterworth M, Alnemri ES, Cohen GM (2001), Recruitment, activation and retention of caspases-9 and -3 by Apaf-1 apoptosome and associated XIAP complexes, EMBO J. 20 998-1009. 

Intrinsic (mitochondrial) pathway of caspase activation is initiated by the permeabilization of the mitochondrial outer membrane by proapoptotic members of the Bcl-2 family, resulting in a release of cytochrome c and other proteins from the intermembrane space of mitochondria into the cytosol. Cytochrome c translocation to the cytosol may follow a number of possible mechanisms. However, once in the cytosol, cytochrome c binds to apoptosis protease activating factor (Apaf-1) and in the presence of dATP or ATP facilitates Apaf-1 oligomerization and the recruitment of procaspase-9. The formation of this caspase-activating complex, termed the apoptosome, results in the activation of procaspase-9, and this in turn cleaves and activates the effector caspase-3 and -7. Activated effector caspases cleave key substrates in the cell and produce the cellular and biochemical events characteristic for apoptosis [33-35]. 

Czerski L, Nunez G. Apoptosome formation and caspase activation Is it different in the heart J Mol Cell Cardiol 2004 37 643-652. 

Cytochrome c 15 kD Electron carrier Component of apoptosome (activation of caspase 9)... 

The most widely studied model is caspase-9 activation. Release of cytochrome c from the mitochondria into the cytosol promotes the assembly of the apoptosome, a complex composed of cytochrome c, Apaf-1 (Apoptosis protease-activating factor-1), and caspase-9. The presence of Apaf-1, which is the specific adaptor for caspase-9, recruits procaspase-9 to the apoptosome resulting in caspase-9 activation (Bao and Shi, 2007 Riedl and Salvesen, 2007). 

It is worth mentioning that in death receptor-mediated apoptosis, cells can be divided into two groups depending on the requirement for mitochondria to induce a complete apoptotic response. Type I cells do not require the mitochondrial pathway because the recruitment of procaspase-8 into the DISC complex is enough to fully activate caspase-8 which then activates effector caspases. However, Type II cells are characterized by an incomplete apoptotic response unless mitochondria are involved (Scaffidi et al., 1999). In this type of cell, efficient activation of effector caspases requires the mitochondrial amplification loop (Fig. 5). Caspase-8 cleaves cytosolic Bid, a BH3-only protein, which when cleaved to tBid is able to translocate to the mitochondria and trigger release of the proapoptotic factors cytochrome c and Smac/DIABLO (Li et al., 1998 Deng et al., 2002). The release of cytochrome c triggers apoptosome formation, subsequent caspase-9 activation, and finally the activation of effector caspases such as caspase-3. 

Zou H, Li Y, Liu X, Wang X (1999) An APAF-1.cytochrome c multimeric complex is a functional apoptosome that activates procaspase-9. J Biol Chem 274 11549-11556 van Loo G, Saelens X, Matthijssens F, Schotte P, Beyaert R, Declercq W, Vandenabeele P (2002) Caspases are not localized in mitochondria during life or death. Cell Death Differ 9 1207-1211... 

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