Apoptosome, formation

Within the nervous system, ChEs were shown to be involved in membrane conductance and transmission of excitatory amino acids, learning and memory, neurite growth, neuritic translocation and acute stress reactions. Recent findings propose AChE s involvement in apoptosome formation [2]. 

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

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. 

The activation of caspase-9 by apoptosome formation sets in motion a cascade of caspase activation events. At the top of this cascade is caspase 3, which cleaves other downstream effector procaspases (caspases-2, -6, -8 and -10) or apoptotic substrates containing the recognition motif DXXD. Activation of this hierarchically structured cascade leads to proteolyis of multiple substrates, and the cell is committed to death. The cellular infrastructure is destroyed, and changes at the plasma membrane are triggered that promote engulfment by phagocytes. 

Fig. 1.3 Cell-death pathways relevant to an apoptotic-like mechanism in cerebral ischemia. Release of cytochrome c from the mitochondria is modulated by pro as well as anti-apoptotic bcl-2 family members. Cytochrome c release activates downstream caspases through apoptosome formation (not shown) and caspase activation can be modulated by smac/DlABLO indirectly through suppressing protein of inhibitors of apoptosis (LAP). Effector caspases (caspase-3, -7) work on multiple substrates which dismantle the cell by cleaving homeostatic,...

Once cyt c has been released into the cytosol, it is able to interact with a protein called Apaf-1 (Figure 3.22). This leads to the recruitment of pro-caspase 9 into a multiprotein complex with cyt c and Apaf-1 called the apoptosome. Formation of the apoptosome leads to activation of caspase cascades, which further leads to activation of apoptosis (Jiang and Wang, 2004). Such cyt c release has been documented for apoptosis induced by chemotherapeutic dmgs, oxidative stress, UV irradiation, serum, glucose deprivation (Kaufmann and Eamshaw, 2000 Kannan and Jain, 2000). 

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. 

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. 

The principal function of cyt. c is to form complexes through a defined interface with protein partners in our cells. This is most established for eukaryotic cytochrome c within the mitochondrial electron transport chain (ETC), a process required for carrying out the oxidative phosphorylation of ATP.4 Formation of a complex with cyt. c reductase (an electron-donor protein from complex III) and cyt. c oxidase (an electron-acceptor protein from complex IV) leads to the transfer of electrons between otherwise separated proteins. More recently cyt. c has been found to play a critical role in the process of apoptosis or programmed cell death This in turn has led to a resurgence of interest in all aspects of cyt. c research.5 Again protein-protein interactions have been shown be essential with mitochrondrial cyt. c binding to such proteins as APAF-1 to form the multi-protein species known as the apoptosome that is now thought to be a requirement for apoptosis.6,7... 

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]. 

Cytochrome C release may follow the MPT or occur independently. In a recent study of dsplatin toxicity [35] decrease in oxidative phosphorylation was due to the inhibition of mitochondrial FO-Fl-ATPase activity, but the decrease in oxidative phosphorylation was accompanied by hyperpolarization of the mitochondrial membrane rather than a decrease in membrane potential that is usually associated with the MPT [36]. The studies also demonstrate a marked decrease in active Na transport and Na-K-ATPase activity that paralleled the decrease in FO-Fl-ATPase activity and preceded increases in membrane potential in cisplatin treated renal proximal tubular cells. These studies would suggest that cytochrome C release into the cytoplasm and the subsequent formation of the apoptosome (see below) may occur independently of the MPT and that the initiation of cell death by disruption of energy metabolism can directly engage the caspase cascade. 

Fig. 15.3 The major pathways of apoptosis. The extrinsic pathway uses extracellular death ligands (Fas ligand, tumor necrosis factor (TNF)) to activate death receptors which pass the apoptotic signal to initiator caspases (e. g. capsase 8) and to the executioner caspases (e. g. caspase 3 caspase 7). In the execution phase of apoptosis, various cellular substrates are degraded leading to cellular collapse. The intrinsic pathway uses the mitochondria as a central component for activation of apoptosis. In this pathway, a multitude of intracellular signals including various stresses, DNA damage and inappropriate cell signaling lead to activation of the pro-apoptotic protein Bax which induces release of cytochrome c from mitochindria, formation of the apoptosome and activation of the initiator caspase 9. Finally, the executioner caspases are activated and cells are destructed by proteolysis. Apoptosis via this pathway can be controlled by various antiapoptotic proteins including the Bcl-2 protein and inhibitors of apoptosis.

In a further step of apoptosis, the cytochrome c released from the mitochondria promotes the assembly of a multiprotein complex, termed apoptosome, which contains cytochrome c, the adaptor protein Apafl, and procaspase-9. The apoptosome requires ATP for its formation and is able to cleave and activate procaspase-3, an effector caspase. The adaptor protein Apafl appears to play a major structural role in this assembly. Apafl contains WD motifs for interaction with cytochrome c and a CARD motif, which directs binding to the CARD motifs of procaspase-9 and procaspase-3. Structural studies on the apoptosome by electron microscopy have revealed a wheel shaped heptameric complex, with the CARD domains of Apafl located at the central hub and the WD40 repeats at the extended spokes (Acehan et al., 2002). The location of pro-caspase 9 in this complex is still open as is the mechanism of caspase 9 activation. 

Depending on the cell type, two different downstream pathways are triggered. In type I cells, processed caspase-8 produced in large amounts directly activates a caspase cascade. Among the caspases activated are caspase-3, which cleaves other caspases or vital substrates of the cell and thus paves the way for the execution phase of apoptosis. In type II cells, proper activation of effector caspases requires amplification via the mitochondrial pathway of apoptosis. Here, smaller amounts of active caspase-3 are produced which cleave the pro-apoptotic Bcl-2 family member Bid. The truncated form of Bid activates mitochondria by an unknown mechanism, which now release pro-apoptotic proteins like cytochrome c and Smac/Diablo (see Section 15.5). Cytochrome c release triggers the formation of the apoptosome, resulting in the activation of caspase-9 and subsequently caspase-3, which in turn can activate caspase-8 outside the Fas-DISC. 

Cytochrome c release is thus an early event during apoptosis, occurring hours before phosphatidylserine exposure and loss of plasma membrane integrity. As mentioned above, it is only after cytochrome c release that caspases areactivated and the cell undergoes apoptosis. The actual apoptotic process occurs through the formation of an apoptosome (comprised of cytochrome c, apoptosis protease activating factor 1 (Apaf-1) and procaspase-9). This apoptosome then recruits procaspase-3, which is cleaved and activated by the active caspase-9 and is subsequently released to mediate apoptosis (Fig. 2) [27]. 

Fig. 18.16. Roles of the Bcl-2 family members in regulating apoptosis. Bcl-2, which is anti-apoptotic, binds Bid (or tBid) and blocks formation of channels that allow cytochrome c release from the mitochondria. Death signals result in activation of a BH3-only protein such as Bid, which can lead to mitochondrial pore formation, swelling, and release of cytochrome c. Bid binds to and activates the membrane ion-channel protein Bax, activating cytochrome c release, which binds to Apaf and leads to formation of the apoptosome.

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