Apoptotic Executioner Caspases

Apoptotic executioner caspases (caspase-3, -6, -7) constitute a subgroup of the caspase family. These proteases are the workhorses of the apoptotic process as they are responsible for cleaving many down-stream substrates important for cellular morphology, organelle homeostasis, cell cycle arrest, and regulation of transcription and translation. 

Apoptotic initiator caspases (caspase-2, -8, -9 and -10) constitute a subgroup of the caspase family. These caspases are the first to become proteolytically active in the apoptotic cascade. Their activation takes place in multiprotein complexes initiated by pro-apoptotic stimuli, such as TNFa, a-Fas, staurosporine. Once activated, they can process their substrates, which include the apoptotic executioner caspases. 

Lippens, S. et al. Epidermal differentiation does not involve the pro apoptotic executioner caspases, but is associated with caspase-14 induction and processing. Cell Death Differ. 7, 1218, 2000. 

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. 

Many elements of complex intrinsic and extrinsic signaling pathways leading to apoptosis have been identified. During apoptosis, a cascade of proteases termed caspases is involved with upstream signaling events and downstream executioner events. Caspases are cysteine-dependent, aspartate-specific proteases that contain highly conserved cysteine residues in their active sites and cleave substrates leaving C terminal Asp residues. Caspase-3 is one of the main effector molecules of the apoptotic process. It cleaves several target proteins and serves as one of the executioner caspases that implement apoptosis. Despite reports of caspase-independent apoptosis (Broker et al. 2005), caspase-3 has become the most widely accepted and most frequently measured apoptosis marker for HTS. 

Effector caspases this group of proteases cleave cellular substrates during apoptosis. Due to their function in the apoptotic paradigm they are also known as executioner caspases. They are characterized by the presence of short prodomains. This group contains caspase-3, -6, and -7. 

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.

At present, there are two relatively well-characterized cell death pathways that result in the activation of executioner caspases (Figure 2). One is receptor-mediated and the other is mitochondrial-dependent. On receiving an apoptotic stimulus, the receptor-depen-dent pathway is initiated by activation of cell death receptors such as Fas and tumor necrosis factor. The death receptor stimulation results in the formation of a death inducing signaling complex (DISC) that recruits and activates procaspase-8, which in turn cleaves and... 

The XIAP-BIR3 domain is responsible for inhibition of the initiator caspase-9, but it functions via a completely different mechanism. The BIR3 domain is an allosteric inhibitor of caspase-9 it binds to the dimer interface and prevents dimerization and subsequent activation of the enzyme (9) (Fig. 4). Caspase-9 is at the apex of the apoptotic cascade that leads to the activation of executioner cascades. As such, BIR3 can provide an extra level of regulation by sequestering monomers in a catalytically inactive conformation and ensuring that no unwanted caspase-9 activity occurs. 

To date, 14 mammalian caspase sequences (named caspases 1 -14) have been reported, of which eleven are of human origin. With respect to function, caspases are grouped into two biologically distinct subfamilies. One subfamily mediates initiation (initiator caspases, caspases-8, -9) or execution (executioner or effector caspases, cas-pases-3, -6 and -7) of the apoptotic program. Members of the other subfamily (cas-pases-1, -4, -5, -11, -12, and -14) are involved in inflammatory processes by processing pro-inflammatory cytokines. 

Apoptosis is a highly conserved cascade of events resnlting in cell destruction without associated inflammation or damage to surrounding tissnes. The executioner enzymes are cysteine-aspartic proteases (caspases) that are normally present as inactive proenzymes (procaspases). Apoptotic stimnli activate initiator caspases (caspase-8, caspase-9, caspase-12) which, in turn, activate effector caspases (caspase-3, caspase-6, caspase-7) that actually dismantle the cell [43]. The apoptotic cascade can be triggered by extrinsic or intrinsic pathways. In the extrinsic pathway, activation of the Fas receptor and tumor necrosis factor receptor (TNF-R) on the cell surface leads to recruitment of adaptor proteins, such as the Fas-associated death domain (FADD), whose death... 

Mechanisms underlying SM-induced apoptosis have been carefully explored using primary cultures of human keratinocytes. Treatment of keratinocytes with 100-300 pM SM resulted in activation of caspase 8, which initiates the Fas-dependent death receptor pathway, and caspase 9, which initiates the mitochondrial apoptotic pathway (Rosenthal et al., 2003). Fas and Fas ligand were upregulated in a concentration-dependent manner by SM leading to activation of caspase 3, the central executioner protease. Transfection of immortalized keratinocytes with a dominant-negative Fas-activated death domain resulted in a blunted caspase response to SM. Micro vesication and tissue injury produced in vivo by SM exposure of transfected cells after grafting onto athymic nude mice was also reduced by this treatment. 

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