Untersuchungen der Ubiquitin-vermittelten Regulation von Apoptose in Drosophila - Ubiquitin-mediated regulation of apoptosis
Zusammenfassung der Projektergebnisse
Activation of caspases, which execute the cell death programme, is an event that has to be tightly regulated. Members of the inhibitor of apoptosis (lAP) protein family ensure cell survival by blocking caspase activity. Ubiquitin-mediated inactivation of caspases has long been postulated to contribute to the regulation of apoptosis. However, detailed mechanisms and functional consequences of caspase ubiquitylation have not been demonstrated. We have been able to show that the Drosophila Inhibitor of Apoptosis 1, DIAP1, blocks effector caspases by targeting them for polyubiquitylation, which depends on DlAP1's own RING finger domain and the recruitment of an Ubiquitin E3 Ligase of the UBR family following N-terminal cleavage. Importantly, this polyubiquitylation does not lead to proteasomal degradation of caspases. Our work demonstrates that the conjugation of Ubiquitin to the Drosophila effector caspase drICE (Caspase 3/7 homologue) significantly suppresses its catalytic potential in cleaving caspase substrates. This data suggest that ubiquitin conjugation sterieally interferes with substrate entry and reduces the proteolytic velocity of the caspase. Moreover, disruption of drlCE ubiquitylation, either by mutation of DIAP1's E3 activity or drlCE's ubiquitin-acceptor lysines, completely abrogates DIAP1's ability to neutralise drICE and suppress apoptosis in vivo. Another surprising discovery was the fact that DIAP1 rests in an 'inactive' conformation and requires caspase-mediated cleavage to become fully competent to bind and ubiqultylate caspases. Increasing levels of active caspases would hence lead to DIAP1 'activation' that is followed by caspase Inhibition. DIAP1's short half-life may ensure that once caspase activity levels subside, the system returns to the resting state In which full-length D1AP1 resides in a 'closed' inactive configuration. In effect, such a scenario would result in a buffered 'oscillating cycle' of caspase activity that ensures the continuous presence of sub-lethal levels of active caspase, which have been shown to be required for various cellular processes. Taken together, the results obtained in this study have strongly enhanced the knowledge of how ubiquitylation is required to control caspase activity and show a new mechanism by which ubiquitylation can regulate protein function directly.
