Caspase regulation: Mechanism of the activation and inhibition of caspase-9

This dissertation discusses the mechanisms of activation and inhibition of caspase-9, an apical caspase central to apoptosis. The structural and biochemical studies entailed herein provide insight into IAP-mediated inhibition of caspase-9 and its activation by apaf-1.; The crystal structure of caspase-9 bound by XIAP-BIR3 reveals the BIR3 fragment disrupts the ability of caspase-9 to dimerize. As such, caspase-9 cannot adopt a conformation conducive to catalysis. Residues important for the recognition of caspase-9 by BIR3 were identified by site-directed mutagenesis, supporting the conclusions drawn from the crystal structure. Based upon these results and contrary to those previously published, it suggests that other members of the IAP family of proteins are unlikely to be capable of inhibiting caspase-9.; Formation of an oligomeric complex reminiscent of the apoptosome/caspase-9 holo-enzyme is found to occur when apaf-1 CARD is added to caspase-9. Concomitant with the formation of the CARD/caspase-9 oligomer is an increase in the catalytic activity of caspase-9. These observations reveal a new function for apaf-1 CARD in the oligomerization of caspase-9 and suggest this oligomer could represent the minimal holo-enzyme.; In an effort to create a dimeric caspase-9 that does not rely on apoptosome activity for such an effect, protein engineering was undertaken. Dimerization of the engineered caspase-9 occurs spontaneously in the absence of additional factors and behaves as a distinct monodispersed species in solution. It is also enzymatically more active than the wild-type protein. However, the activity of the engineered dimer is significantly below that of the wild-type caspase-9 bound to the apoptosome. Interestingly, the activity of the engineered dimer is unaffected by the presence of the apoptosome. These results indicate that apoptosome-mediated activation of caspase-9 entails a mechanism more elaborate than just dimerization. A body of biochemical evidence has been accumulated and has allowed a working model for the activation of caspase-9 to be proposed.