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Caspases are cysteine proteases that are key effectors in apoptotic cell death. Currently, there is a lack of tools that can be used to monitor the regulation of specific caspases in the context of distinct apoptotic programs. We describe the development of highly selective inhibitors and active site probes and their applications to directly monitor executioner (caspase-3 and -7) and initiator (caspase-8 and -9) caspase activity. Specifically, these reagents were used to dissect the kinetics of caspase activation upon stimulation of apoptosis in cell-free extracts and intact cells. These studies identified a full-length caspase-7 intermediate that becomes catalytically activated early in the pathway and whose further processing is mediated by mature executioner caspases rather than initiator caspases. This form also shows distinct inhibitor sensitivity compared to processed caspase-7. Our data suggest that caspase-7 activation proceeds through a previously uncharacterized intermediate that is formed without cleavage of the intact zymogen.

Caspase-7 is an obligate dimer of catalytic domains, with generation of activity requiring limited proteolysis within a region that separates the large and small chains of each domain. Using hybrid dimers we distinguish the relative contribution of each domain to catalysis by the whole molecule. We demonstrate that the zymogen arises from direct dimerization and not domain swapping. In contrast to previous conclusions, we show that only one of the catalytic domains must be proteolyzed to enable activation. The processed domain of this singly cleaved zymogen has the same catalytic activity as a domain of fully active caspase-7. A transient intermediate of singly cleaved dimeric caspase-7 can be found in a cell-free model of apoptosis induction. However, we see no evidence for an analogous intermediate of the related executioner caspase-3. Our study demonstrates the efficiency by which the executioner caspases are activated in vivo.