Using the DNA sequence encoding the active site of caspase-1 and ced-3 to search an expressed sequence tag (est) database, a human sequence was identified, cloned and shown to encode a 32 kDa cysteine protease, called CPP32 (Fernandes Alnemri et al., 1994). Independently, two other groups identified caspase-3, one naming it Yama (the Hindu god of death) and the other apopain (Tewari et al., 1995; Nicholson et al., 1995). Caspase-3, is widely distributed, with high expression in cell lines of lymphocytic origin, suggesting that it may be an important mediator of apoptosis in the immune system (Fernandes Alnemri et al., 1994). Based on the cleavage site of PARP (DEVD¯ G), Ac-DEVD-AMC was synthesised as a model substrate, and Ac-DEVD.CHO and its biotinylated derivatives were synthesised as specific inhibitors of PARP cleavage and as affinity ligands for purification of the protease. Using electrospray MS and N-terminal sequence analysis, the acive enzyme was shown to be composed of two subunits of 17 kDa and 12 kDa, derived from the precursor protein by cleavage at Asp-28-Ser-29 and Asp-175-Ser-175 (Nicholson et al., 1995). While the initial cleavage is probably between the large and the small subunit, it has been suggested that processing within the prodomain occurs initially at Asp-9, not at Asp-28 (Fernandes-Alnemri et al., 1996; Srinivasula et al., 1996).
The three-dimensional structure of a complex of caspase-3 with DEVD.CHO shows that, although caspase-3 resembles caspase-1 in overall structure, its S4 subsite is very different in size and chemical composition and accounts for their differences in specificity (Rotonda et al., 1996). The S4 subsite of caspase-1 is a large shallow hydrophobic depression that readily accommodates a tyrosyl side chain, while the analogous site in caspase-3 is a narrow pocket that closely surrounds the P4 Asp side chain (Rotonda et al., 1996). The Trp residue at position 348 and an inserted 10-amino-acid sequence at position 381 play a crucial role in defining size and shape of the S4 subsite of caspase-3 and are also conserved in all known caspases. The activation of caspase-3 to either of its catalytic active p17 or p12 subunits has been demonstrated in cells undergoing apoptosis.
Caspase-3 is one of the key executioners of apoptosis, being responsible either totally or partially for the proteolytic cleavage of many key proteins, each of which contains a common Asp-Xaa-Xaa-Asp (DXXD) motif, similar to the one originally described in poly(ADP-ribose) polymerase PARP (O'Shaughnessy et al., 1994).
It was the discovery of this substrate, that provided a direct link to a lack of DNA repair (which might explain the formation of the DNA ladders during apoptosis) and made caspase-3 right from the start a "killer-gene" (Lazebnik et al., 1994). These findings however are contradicted by recent literature and the healthy phenotype of the PARP knock-out mice. Today it is clear the PARP cleavage and the process apoptosis are not explicitly on identical pathways, so one can have apoptosis without PARP cleavage and vice versa.
Caspase-3 -/- mice, generated by homologous recombination, are smaller than their littermates and die at 1-3 weeks of age. Thymocytes from caspase-3-deficient mice show a similar sensitivity to apoptosis induced by a number of different stimuli including CD95, anti-CD3, staurosporine and dexamethasone. Brain development in these deficient mice is markedly affected, with a variety of hyperplasias being observed from embryonic day 12. Pyknotic clusters of apoptotic cells, observed at sites of major morhogenetic change in normal brain development, are not seen in the deficient mice, indicative of decreased apoptosis in the absence of caspase-3. This demonstrates that caspase-3 plays a critical role during morphogenetic cell death in the mammalian brain. The restricted phenotype also raises the possibility that other caspases may be important in other tissues or cell types (Kuida et al., 1996).