Characterisation of de-ubiquitylating enzymes in the regulation of apoptosis

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In Drosophila, the regulation of cell survival depends critically on the Inhibitor of Apoptosis (IAP) protein DIAP1. Loss of DIAP1 leads to immediate caspase activation and apoptotic cell death. DIAP1 regulates the initiator caspase DRONC (homologue of caspase-9) and the effector caspases drICE and Dcp-1 (common homologues of caspase-3 and -7). For this purpose, DIAP1 requires a RING domain, providing it with E3-ligase activity for ubiquitin and the ubiquitin-like protein Nedd8.Attachment of the small modifier ubiquitin to lysine residues of the target protein can have various effects. Ubiquitin cannot only be attached as a single unit (mono-ubiquitylation) but can also form chains (poly-ubiquitylation) through internal lysine (K) residues in its own sequence. The linkage point (7 lysines present within the ubiquitin sequence) determines largely the consequence of the ubiquitylation. The best-studied chain types are K48-linked chains, which lead to proteasomal degradation of the ubiquitylated protein, and K63-linked chains, which mainly lead to formation of protein complexes and signalling events. To remove ubiquitin from proteins, de-ubiquitylating enzymes (DUBs) have evolved. DIAP1-mediated ubiquitylation of caspases has been shown to contribute to DIAP1s anti-apoptotic potential. We and others have demonstrated that DIAP1 ubiquitylates the initiator caspase DRONC and the effector caspases Dcp-1 and drICE. Recent data indicate non-degradative mechanisms by which the caspases get inactivated, rather than ubiquitin-mediated proteasomal degradation. However, under conditions where caspases have to be activated, these modifications need to be removed to allow caspase activation. To identify de-ubiquitylating enzymes that are involved in the regulation of apoptosis, we have performed an in vivo RNAi screen. To this end, RNAi fly lines of all predicted Drosophila de-conjugating enzymes were expressed in the developing eye of flies in combination with the proapoptotic IAP-antagonists reaper (rpr) and head involution defective (hid). This led to the identification of enzymes that, when knocked-down, either suppress or enhance the IAP-antagonist induced small eye phenotype. In this study, we plan to characterise two hits of this in vivo screen to elucidate the role of de-ubiquitylating enzymes in the regulation of apoptosis. This will lead to the identification of new mechanisms that determine whether a cell lives or dies.