Apoptosis protease-activating factor

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. 

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]. 

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). 

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]. 

Cytochrome c-dependent mitochondrial apoptosis involves a number of concerted steps leading to the formation of the apoptosome and the downstream activation of caspase-3 [10,11]. The predominant mechanism is believed to involve the following events (Fig. 4) (1) a transient collapse of the mitochondrial transmembrane potential (2) opening of the permeabiUty transition pore (FTP) (3) release of cytochrome c and procaspase-9 (4) recruitment of Apaf-1 (apoptosis protease activating factor-1) and dATP and assembly of the mitochondrial apoptosome (5) activation of procaspase-9 by the apoptosome and (6) the caspase-9-catalyzed activation of caspase-3, the molecular executor of apoptosis. Amplification through the caspase cascade leads to the downstream... 

It is now well estahlished that activation of the caspase cascade is an indispensable and sufficient process in the execution phase of apoptosis (Nunez et al, 1998). As for mitochondria-mediated apoptosis, cytochrome c released from the mitochondrial inner membrane is well known to play an important role in the activation of caspase 9, one of the upstream proteases in the cascade (Zou et al, 1997). For activation of caspase 9, cytochrome c or apoptotic protease activating factor 2 (Apaf 2) induces the formation of the complex between Apaf 1 and caspase 9. The resultant activated caspase 9 then activates caspase 3, which in turn leads to the genomic DNA fragmentation and apoptotic cell death. 

Fig. 1. Proposed mechanism of action of rituximab associated with the apoptosis pathway. Binding of rituximab with the CD20 antigen up-regulates the production of interleukin-10 (IL-10). The IL-10 autocrine loop down-regulates the expression of the bcl-2 protein, which inhibits the intrinsic pathway (or mitochondrial mediated pathway) of apoptosis. The mitochondrial pathway is induced by intracellular stress signals. The translocation of the bcl-2 protein into the mitochondria leads to the activation of caspase 9 via release of cytochrome c and apoptotic protease-activating factor 1. The other pathway, the extrinsic pathway (or death receptor mediated pathway) activates caspase 8. Subsequently, caspase 8 or 9 activates caspase 3, leading to programmed cell death (apoptosis).

The activation of the inflammatory caspases uses a mechanism resembling that of the initiator caspases. The presence of a complex, known as the inflammasome (Martinon et al., 2002), is required for activation of this set of proteases. The recruitment of caspases into this complex results in their activation. For caspase-1, the adaptor ASC (apoptosis-associated specklike protein containing a CARD) is critical in inflammasome formation in response to a variety of stimuli, whereas involvement of the adaptors Ipaf (ICE-protease-activating factor) and NALP3 is stimulus-dependent (Mariathasan, 2007). 

Sustained elevations in intracellular Ca levels promote mitochondrial Ca uptake, which decreases the mitochondrial membrane potential and blocks the electron transport chain leading to ATP depletion. Large increases in mitochondrial Ca uptake also increase mitochondrial membrane permeability (Dubinsky and Levi, 1998) resulting in the release of proapoptotic factors such as cytochrome c and apoptosis inducing factor (AIL) (Luetjens et al, 2000). Cytochrome c binds to apoptotic protease-activation factor 1 (APAFl) and procaspase-9, forming a multiprotein complex known as the apoptosome that activates the caspase cascade. The apoptosome activates caspase-9, which in turn activates caspase-3. Caspase-3 activates caspase-activated DNase (CAD), resulting in DNA fragmentation, characteristic of apoptosis. 

Apoptotic protease activation factor-1, a cytosolic protein involved in cell death or apoptosis, interacts with cytochrome c to activate caspase 9... 

Fig. 1. Modulation of apoptosis by v-FLIP and v-Bcl-2. v-FLIPs specifically inhibit apoptosis mediated by death receptors. v-lCA specifically targets caspase-8 and inhibits its activation. v-Bcl-2 and vMIA inhibit those apoptotic pathways that are signaled through mitochondrial release of cytochrome c. FADD, Fas-associated death domain FLICE, FADD-like interleukin-converting enzyme CARD, cas-pase-recruiting domain PTPC. permeability transition pore complex FLIP, FLICE-inhibitory protein vie A, viral inhibitor of caspase 8-induced-apoptosis MIA. viral mitochondrial inhibitor of apoptosis Apcif-l, apoptotic protease-activating factor 1...

Interestingly, evidence has been obtained that Fas may induce apoptosis via different pathways that are used almost exclusively in different cells (Scaffidi et al. 1998). In some cells. Fas ligation induced a rapid and strong activation of caspase-8 and caspase-3 (see above and Fig. 1) and subsequent apoptosis was not blocked by Bcl-2. In other cells, Fas-ligation resulted in a considerably weaker activation of FLICE (caspase-8), which cleaves Bid, a proapoptotic member of the Bcl-2 family (Luo et al. 1998 Li et al. 1998). Cleaved Bid subsequently translocates to mitochondria, where it promotes, either alone or together with Bax, cytochrome c efflux (Eskes et al. 2000). Subsequently, cytochrome c can interact with the cytosolic apoptotic protease-activating factor 1 (Apaf-1), which in turn activates caspase 9... 

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