Systematic in vivo RNAi analysis identifies IAPs as NEDD8-E3 ligases

Area of Science:

• Cell Biology
• Molecular Biology
• Biochemistry

Background:

• The ubiquitin (Ub)-signaling system is crucial for cell death and survival, but the roles of other Ub-like proteins (UBLs) and their modifying enzymes are less understood.
• While ubiquitylation is well-studied, the impact of NEDD8 conjugation and deconjugation on cellular processes, particularly apoptosis, remains largely unexplored.

Purpose of the Study:

• To investigate the role of NEDD8 conjugation and deconjugation in regulating apoptosis.
• To identify novel regulators of cell death pathways involving Ub-like proteins.

Main Methods:

• Systematic in vivo RNAi screening to identify genes suppressing apoptosis upon knockdown.
• Genetic ablation of deneddylase 1 (DEN1) to study its role in apoptosis.
• Investigating the E3 ligase activity of inhibitor of apoptosis (IAP) proteins in the NEDD8 conjugation pathway.
• Assessing the effect of DEN1 on NEDD8 modification of effector caspases.

Main Results:

• Knockdown of three NEDD8-specific isopeptidases suppressed apoptosis, suggesting a pro-apoptotic role for NEDD8 removal.
• Genetic ablation of DEN1, a key deconjugating enzyme, also suppressed apoptosis.
• Drosophila and human IAP proteins were identified as E3 ligases that target effector caspases for neddylation and inactivation.
• DEN1 was shown to reverse the inhibitory effect of NEDD8 attachment by removing the NEDD8 modification from effector caspases.

Conclusions:

• The study reveals that NEDD8 conjugation, mediated by IAPs, acts as a novel mechanism to inhibit effector caspases and prevent apoptosis.
• DEN1 plays a critical role in reversing NEDD8-mediated inhibition of apoptosis.
• These findings expand the known functions of IAPs beyond ubiquitylation, highlighting their involvement in NEDD8 conjugation and extending the complexity of IAP-mediated signaling in cell death regulation.