Identifying new cell death regulators.

Despite the identification of numerous components of the cell death machinery, little was known about their physiological role. To this end, we have studied the requirement of all Drosophila caspases (7) and caspase-adaptors (2) as well as the second Drosophila IAP, DIAP2, in different paradigms of developmentally regulated and stress induced apoptosis.

Of the seven caspases, Dronc, drICE, Strica and Decay were found to be rate-limiting for apoptosis. Surprisingly, Hid-mediated apoptosis required a broader range of caspases than apoptosis initiated by loss of the caspase-inhibitor DIAP1 suggesting that Hid causes apoptosis not only by antagonising DIAP1 but also by activating DIAP1-independent caspase cascades.

Furthermore, we found that overexpression of the second Drosophila IAP DIAP2 can rescue diap1-RNAi-mediated apoptosis, indicating that DIAP2 regulates caspases directly. Consistently, we found that DIAP2 binds active drICE. Animals that lack DIAP2 harbour significantly increased levels of drICE activity. In agreement with higher levels of active caspases, diap2 mutant cells are sensitised to apoptosis following exposure to sub-lethal doses of X-ray irradiation. Our molecular characterisation has uncovered an unexpected mechanism through which DIAP2 restrains the target enzyme drICE. Surprisingly, DIAP2 acts as a pseudo-substrate that, following cleavage, seem to trap the active caspase via a covalent linkage between DIAP2 and the catalytic machinery of drICE. In addition, DIAP2 also requires a functional RING finger domain to block cell death, and to target drICE for ubiquitylation. Since DIAP2 efficiently interacts with drICE, our data suggest that DIAP2 controls drICE in its apoptotic and non-apoptotic roles. Currently, we are examining the role of DIAP2 in regulating drICE in its non-apoptotic roles.