Caspase protease activity

Recently, a SAMDI-MS assay was described by means of which endogenous caspase protease activities in cell lysates can be determined [26], Similar to the assay used to determine anthrax lethal factor inhibitors, peptide substrate SAMs for either caspase-3 or -8 were treated with cell lysates. In contrast to fluorescence assays, also longer peptide substrates could be used, thus enabling a better resolution of the two caspase activities. 

Apoptosis (programmed cell death) is characterized by a complex series of biochemical changes that culminate in cell death without inflammation or swelling, which are signs of necrosis. Embryonic, fetal, and postnatal development involve cell death by apoptosis, which serves to eliminate excessive cell proliferation and migration. Apoptosis is initiated by a variety of external stimuli and molecular events such as oxidative stress, mitochondrial permeability transition, mitochondrial cytochrome c release, activation of caspase proteases, activation of endonucleases, transglutaminase activation, and poly(ADP-ribose) polymerase cleavage. 

Various fluorescence based kits, e.g. those using annexin V conjugates Caspase protease activity... 

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. 

Like many other proteases, the caspases are formed as inactive proenzymes of 30—50 kDa and are activated by proteolytic processing. The proenzymes have a prodomain and two cleavage sites for processing, which are consensus sites for the caspases, enabling activating by autoproteolysis. 

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).

When caspase 8, an initiator caspase, is activated by an apoptotic signal carried through FADD, it further self-activates by cleaving its own proenzyme form. Mitochondria are one target of active caspase 8. The protease causes the release of certain proteins contained between the inner and outer mitochondrial membranes ... 

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. 

Effector caspases are activated by a transactivation mechanism, which is characterized by the catalytic action of a mature caspase on a procaspase (Thornberry et al., 1997 Earnshaw et al., 1999 Slee et al., 1999). Nevertheless, their activation can also occur by the action of other proteases. Granzyme B, a serine-protease, also has proteolytic specificity for aspartic acid residues. It is able to cleave and directly activate caspase 3 (Darmon et al., 1995). Cathepsin B, a lysosomal protease, cleaves and activates procaspase 11 (Schotte et al., 1998). 

Figure 7.11 Activation of the caspase proteases during apoptosis. Caspases are implicated in both the induction and execution of the apoptotic process. Following the apoptotic stimuli, initiator caspases (caspase 8 or 9) are activated by autocatalysis. The initiator caspases then activate the effector caspases (caspase 3, 6, and 7), which are responsible for most of the protein cleavage during apoptosis.

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]. 

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... 

Thomson, R.H., 1971, Naturally occurring quinones. Academic Press, New York Vancompemolle, K., van Herreweghe, F., Pynaert, G., van de Craen, M., De Vos, K., Totty, N., Sterling, A., Fiers, W., Vandenabeele, P., and Grooten, J., 1998, Atractyloside-induced release of cathepsin B, a protease with caspase-processing activity. FEBSLett, 438 ISO-158... 

Signalling for apoptosis can involve a plasma Fas ligand which binds to the PM Fas receptor with resultant activation of an associated cytosol-side Fas death domain of Fas and activation of caspase 8. Caspase 8 is a thiol protease and once activated initiates a so-called caspase cascade leading to activation of further caspases (with consequent proteolysis) and activation of a DNase (leading to DNA destruction with formation of a characteristic DNA fragment ladder ). Caspase 8 acts on mitochondria with resultant release of cytochrome c, which promotes caspase 3 activation by caspase 8 and hence the caspase cascade . Another signalling pathway for apoptosis involves tumour necrosis factor (TNF) binding to the TNF receptor with consequent activation of a cytosolic-side TNF receptor-associated death domain (TRADD) and resultant activation of the caspase cascade and cell death. 

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 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). 

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). 

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