Apoptosis Protease activation

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... 

YU R, mandlekar s, HARVEY K J, UCKER D s and KONG A N (1998) Chemopreveutive isothiocyanates induce apoptosis and caspase-3-like protease activity . Cancer Res, 58 402-8. 

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. 

P. Kwo, T. Patel, S. F. Bronk and G. J. Gores, Nuclear serine protease activity contributes to bile-acid-induced apoptosis in hepatocytes. Am. J. Physiol, 1995, 268, G613. 

During apoptosis, the mitochondrial permeability is altered and apoptosis-specific protease activators are released from this organelle. The discontinuity of the outer mitochondrial membrane results in the release of cytochrome C to the cytosol followed by subsequent depolarization of the inner mitochondrial membrane (C5, PI). The release of cytochrome C further promotes activation of cas-pases, which are important molecules for initiating apoptosis (T6). Apoptosis inducing factor (AIF), another molecule released into the cytoplasm, has proteolytic activity and is by itself sufficient to induce apoptosis. 

H7. Hirsch, T., Marchetti, R, Susin, S. A., Dallaporta, B., Zamzami, N., Marzo, L, Geuskens, M., and Kroemer, G., The apoptosis-necrosis paradox. Apoptogenic proteases activated after mitochondrial permeability transition determine the mode of cell death. Oncogene 15, 1573-1581 (1997). 

M5. Martin, S. J., and Green, D. R., Protease activation dtrring apoptosis Death by a thousand cuts Cell 82, 349-352 (1995). 

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).

Induction of apoptosis associated with increased caspase (cysteine protease) activity and decreased expression of the anti-apoptotic Bcl-2 protein along with increased expression of the pro-apoptotic Bak and Bax proteins... 

Caspases are involved in intracellular proteolytic protease activation cascades leading to apoptosis that are initiated by ligands such as tumour necrosis factor (TNF) and Fas ligand. These proteins bind to PM receptors with cytosolic death domains that activate the cas-pase cascades leading to cell death. Caspases are cysteine proteases that cleave peptide bonds on the carboxyl side of aspartate (hence c-asp-ases). 

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). 

Verhagen AM, SUke J, Ekert PG, Pakusch M, Kaufmann H, Connolly LM, Day CL, Tikoo A, Burke R, Wrobel C, Moritz RL, Simpson RJ, Vaux DL (2002) HtrA2 promotes cell death through its serine protease activity and its ability to antagonize inhibitor of apoptosis proteins. J Biol Chem 277 445 54... 

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. 

Verhagen AM, et al. HtrA2 promotes cell death through its serine protease activity and its ability to antagonize inhibitor of apoptosis proteins. J. Biol. Chem. 2002 277 445-454. 

---