Caspases are highly specific cysteine-aspartate proteases with well-documented functions in inflammation and apoptosis. Most of the twelve human caspases have been well characterised, however the roles of some still remain elusive. This is the case for caspase-2.
Caspase-2 was the second identified human caspase. Of all the human caspases, it has the highest sequence homology to Ced-3, the only C.elegans apoptotic caspase. This evolutionary conservation implies a significant function for caspase-2.
Caspase substrates illustrate the function of the caspase; therefore it was hypothesised that identifying novel caspase-2 substrates may elucidate its function. This study initially sought out to determine a caspase-2 cleavage-site sequence consensus. This would be highly informative because a specific consensus unique to caspase-2 had not previously been found.
Using a unique yeast-based reporter system, we determined that an XDTTD’G cleavage site is most efficiently proteolysed by caspase-2. Experiments using fluorogenic peptides showed that caspase-2 cleaved VDTTD more efficiently than VDVAD, which is acclaimed to be the classical caspase-2-cleavable peptide. As well, VDTTD activity was more specific for caspase-2 than VDVAD was, since caspase-3 exhibited very little activity against it but cleaved VDVAD better than caspase-2. Furthermore, in vitro experiments using wild-type and mutant forms of Bid, a Bcl-2 relative previously identified as a caspase-2 substrate, showed that a mutant bearing the sequence DTTD’G rather than the natural cleavage site (LQTD’G) was more efficiently cleaved by caspase-2 than the wild-type substrate.
Using the specificity data generated from the yeast reporter system, a model was generated using a bioinformatics program, Prediction of Protease Specificity (PoPS), and potential novel substrates for caspase-2 were identified. One of the highest-scoring candidates, Runx1, was subsequently proteolysed in vitro by caspase-2 at the site predicted by PoPS. Further experiments are currently in progress to determine if this cleavage event occurs in cells.