Towards a molecular mechanism

Due to the widespread importance of apoptosis in many biological processes there has been intense interest in understanding how this process works at a molecular level. By 1993, although much of the cell biology of apoptosis had been established and several key apoptosis regulatory molecules, such as Bcl-2 and p53, had been discovered, very little was known concerning the nature of the cell death machinery itself. In 1993 a major breakthrough occurred when it was reported that CED-3, one of the genes that was known to be required for all the developmental-related programmed cell deaths in the nematode worm Caenorhabditis elegans, was homologous to ICE (caspase-1). ICE is a cysteine protease that has been implicated in the processing of IL-ip to its mature form and is unusual as it was the first protease described that cleaves after aspartate (Asp) residues. Recent studies suggest that ICE itself probably does not play a pivotal role in apoptosis: rather several of its mammalian homologs (including CPP32 (caspase-3), Mch2 (caspase-6), Mch3 (caspase-7) and mch5 (FLICE/Caspase-8)) are involved in the cell death signal. These caspases, which cleave after Asp residues, appear to be constitutively expressed in most cells as inactive precursors that require proteolytic processing to achieve their active forms (Figure 4).

Evidence that proteases are central to apoptosis

The evidence that proteases may be key components of the cell death machinery is derived from several lines of investigation. Early indications that proteases may participate in apoptosis was suggested by observations that several proteins underwent proteolytic cleavage during this process. Additional evidence was provided by the discovery that certain broad-spectrum protease inhibitors could block or delay apoptosis. Strong support for a role for caspases in apoptosis has subsequently been provided by studies that have examined the effects on apoptosis of CrmA, a cowpox virus-derived serpin (serine protease inhibitor) that was known for its ability to act as a pseudosubstrate for, and thereby neutralize, ICE. Several groups have independently reported that CrmA potently blocks several forms of apoptosis, including that induced by ligation of the CD95 molecule (Fas/APO-1) as well as due to CTL attack. Another pseudosubstrate for caspases was recently discovered in the form of the baculovirus

Prodomain p20

Inactive ICE I proenzyme [

'Convertase'

Active ICE

Figure 4 Caspase (ICE/CED-3) family proteases require proteolytic processing to achieve their mature forms. Most caspases are probably constitutively expressed as inactive precursor proteins that require further proteolytic processing at Asp residues in order to achieve their active heterodimer forms (that may further associate to form dimers of heterodimers). Maturation of some caspases is seen during the early stages of apoptosis (CPP32 and Mch3, for example).

p35 protein, which was already known for its ability to act as a potent repressor of apoptosis.

Many recent studies have also found that specific peptide inhibitors of caspases (YVAD, DEVD, DEAD, VAD) inhibit many forms of apoptosis. All of this evidence for protease involvement in apoptosis is supported by the ongoing discovery of new proteins that are cleaved during this process. Together these data heavily implicate proteases as central components of the cell death machinery and suggest that the caspases play a critical role in apoptosis (Figure 5).

How protease activation may lead to the apoptotic phenotype

So how do proteases produce the apoptotic phenotype in a cell? The short answer is that we do not know as yet. Some informed guesses can be made by looking at the nature of the substrates that are cleaved during apoptosis. To date, most of the substrates that have been documented to undergo proteolytic cleavage during apoptosis are localized in the nucleus. It is likely that many of these cleavage events are not contributory to the apoptotic phenotype but are merely bystander effects due to these proteins possessing the appropriate cleavage sites. This idea is strongly supported by the observation that enucleated cells can also undergo apoptosis. However, it is relatively easy to conceive how cleavage of some of these proteins may directly lead to changes in the cell that frequently occur during apoptosis. For example, cleavage of the nuclear lamins, proteins that are largely responsible for the maintenance of the integrity of the nuclear envelope, could be directly responsible for the collapse (condensation) of the nucleus that is seen during apoptosis. Proteolysis of fodrin, a cytoskeletal protein that plays a major role in the cortical cytoskeleton, could lead to the plasma membrane blebbing that is typical of apoptosis (Figure 3). Proteases could also serve to degrade an inhibitor of the endonuclease that cleaves DNA at internucleosomal sites during apoptosis. Indeed, a protease with nuclease-activating properties has been described. Further studies are required in order to fully investigate the impact of each of the cleavage events upon cell integrity and to elucidate how they impact upon the cell death process.

Figure 4 Caspase (ICE/CED-3) family proteases require proteolytic processing to achieve their mature forms. Most caspases are probably constitutively expressed as inactive precursor proteins that require further proteolytic processing at Asp residues in order to achieve their active heterodimer forms (that may further associate to form dimers of heterodimers). Maturation of some caspases is seen during the early stages of apoptosis (CPP32 and Mch3, for example).

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