Investigating the molecular mechanism through which IAPs block caspases

The covalent attachment of Ub to target proteins can alter their conformation and/or binding properties changing its activity, localisation or half-life. IAP-mediated mono- and poly-ubiquitylation of caspases has been reported for a number of IAPs and caspases; however, biochemical insights into the actual molecular and functional consequences of caspase ubiquitylation are lacking.

Using Drosophila as a model system, we have addressed the molecular mechanism and functional consequence of IAP-mediated ubiquitylation of effector caspases. The Drosophila IAP DIAP1 is the key anti-apoptotic molecule in Drosophila that safe-guards cell viability by neutralising the initiator caspase Dronc (the Drosophila orthologue of mammalian caspase-9) and the effector caspase drICE and DCP-1 (caspase-3 in mammals). While it is clear that DIAP1 regulates cell death through its ability to function as an E3 Ub-protein ligase, it remained enigmatic how Ub conjugation impacts on caspase activity. We have now identified how the E3 Ub-protein ligase activity of DIAP1 neutralises caspases. We find that DIAP1 promotes polyubiquitylation of drICE and DCP-1 in a N-end rule and RING finger dependent manner. Surprisingly, polyubiquitylation of drICE and DCP-1 does not target these caspases for proteasomal destruction. Instead, we find that polyubiquitylation of active effector caspases leads to their non-degradative inactivation. Most likely, the conjugation of Ub to drICE results in steric interference with substrate entry and, in addition, allosteric conformational impairment of the caspase's catalytic pocket. In this respect, ubiquitylation acts as a 'mixed' inhibitor displaying both competitive (increased KM) and non-competitive (decreased Vmax) inhibitory properties. Consistent with the notion that caspase ubiquitylation leads to the inhibition of the enzymatic activity, we find that a drICE K>R mutant, which can no longer be ubiquitylated, is fully resistant to DIAP1-mediated inhibition. Together, our results demonstrate that polyubiquitylation of caspases lead to their inactivation in a degradation-independent manner. This observation has important implications for caspase activation, as it predicts that de-ubiquitylating enzymes (DUBs) can ‘reactivate’ ubiquitylated caspases.

Ub-mediated regulation of caspases

Moreover, we find that full-length DIAP1 rests in an ‘inactive’ configuration that prevents caspase-binding and suppresses its E3 Ub-ligase activity. DIAP1’s anti-apoptotic activity is activated by caspase-mediated cleavage, providing the cell with a sensitive strategy to monitor and neutralise active caspases. Almost 300 million years of evolutionarily selection pressure has preserved a caspase cleavage site in insect IAPs that, following processing by a caspase, exposes a binding motif for the E3 Ub-ligases of the N-end rule pathway (UBR-E3s). Removal of DIAP1’s N-peptide is also required for caspase binding. Thus, recruitment of the UBR-E3 ubiquitylation-machinery into the ‘cleaved-IAP::caspase’-complex provides a mechanism to regulate effector-caspases in a negative feedback loop. Our data provides a likely explanation how cells maintain buffered levels of active caspases without losing cell viability. It appears that caspases, through cleaving DIAP1, initiate their own inhibition. Such a scenario would result in a buffered ‘oscillating cycle’ of caspase activity that would establish a continuous presence of sub-lethal levels of active caspases.

Other projects