Generic cell death cysteine proteases are activated by graB cleavage

Studies in the helminth Caenorhabditis elegans have elucidated a key cysteine protease, CED-3 with Asp-ase specificity that is essential for the death of 131 somatic cells programed to undergo apoptosis during normal embrogenesis. The first mammalian homolog of CED-3 recognized was interleukin Ip-converting enzyme (ICE), which was initially identified for its cleavage (at Asp residues) and activation of IL-1. All the caspases (ICE-like proteases) are synthesized as proenzymes which require cleavage at a specific Asp residue to produce an active heterodimeric molecule. Further autoprocessing then results in cleavage (also at Asp) of the propeptide, resulting in the fully active mature form. Expression of active ICE in a variety of cell lines resulted in apoptotic cell death. Subsequently, three subfamilies of related cell death proteases have been identified, whose individual members share closest structural similarity to either CED-3, ICE or ICH-1 (Nedd-2). It is the current view that some or all of these proteases are expressed in an inactive form in virtually every mammalian cell, and can be activated in response to a very diverse range of apoptotic stimuli including toxins, irradiation, growth factor withdrawal and intracellular pathogens.

That the Asp-ase activity of graB was indispensable for perforin/graB-mediated cell death strongly suggested it might activate ubiquitous cysteine protease cell death pathways. The lack of requirement for protein or RNA synthesis in CTL/NK-mediated apoptosis is consistent with latent cytoplasmic ligands/substrates 'ready' for cleavage by graB. Strong evidence now exists that graB can cleave multiple cell death proteases of the CPP32 (CED-3-like) subfamily that constitute a protease cascade in the cytoplasm of target cells (Figure 1). CPP32 (also termed apopain and YAMA-1) activation by cleavage at Aspl75 is followed by autoprocessing at Asp9, resulting in the fully active 19/12 kDa form that can cleave nuclear structures such as poly-(ADP ribose) polymerase (PARP). Cleavage (and inactivation) of PARP and other enzymes including DNA-dependent protein kinase (DNA-PK) has been identified in nuclei early in the apoptotic event. This can result in curtailed DNA repair and derepression of nucleases that then cleave exposed internucleoso-mal sites on DNA, contributing to DNA degradation and precipitating nuclear breakdown. In addition, graB can activate cytoplasmic proMch4 which in turn cleaves proCPP32 and proMch3, further amplifying the proteolytic cascade. Activation of Mch2 then results in the proteolysis of structural nuclear proteins such as lamins.

Evidence also exists that some graB substrates, such as proMch4 and proMch5 (ELICE), are high in the proteolytic cascade, and may also be able to couple Fas ligation to downstream proteolytic events. This is achieved in that proMch4 and proMch5 both contain two N-terminal FADD-likc domains. These regions may associate with FADD bound to the cytoplasmic domain of Eas following FasL binding and receptor aggregation, and lead to activation of the cysteine protease domains of Mch4 and Mch5 to trigger downstream events in the cysteine protease cascade. Thus, in some instances CTLs may be able to activate proMch4/5 via Fas, and in other instances may use graB to activate them directly.

Remarkably, some viruses have developed ways of subverting generic cell death pathways by carrying protease inhibitors (serpins) whose expression can subvert 'altruistic' apoptosis of a cell, leading to a prolongation of the cell's life and greater time for viral replication and assembly. For example, the protein encoded by the cowpox crmA gene can block ICE activation and prevent apoptosis, while the baculovirus protein p35 can derail apoptotic mechanisms by a similar process. Serpins may also inhibit both cysteine and serine proteases of like-spccificitv (cross class inhibition). It is interesting that crmA is far more effective at blocking ICE than graB, suggesting that cells in which ICE activation has been blocked might still be susceptible to CTL/NK-mediated apoptosis due to graB.

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