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Published on: October 6, 2022
Interactions of DNR1 with the apoptotic machinery of Drosophila melanogaster
David A Primrose1, Sidharth Chaudhry, A George D Johnson
1Department of Medical Microbiology and Immunology, University of Alberta, Edmonton AB, T6G 2S2, Canada.
Abstract:
Caspases are crucial activators of apoptosis and NF-kappaB signaling in vertebrates and invertebrates. In Drosophila, the caspase-9 counterpart Dronc is essential for most apoptotic death, whereas the caspase-8 homolog Dredd activates NF-kappaB signaling in response to gram-negative bacterial infection. The mechanics of caspase regulation are conserved and include the activities of a family of inhibitor of apoptosis (IAP) proteins. The RING-domain-bearing protein Defense repressor 1 (Dnr1), blocks ectopic Dredd-mediated induction of an NF-kappaB reporter in the Drosophila S2 cell line. In this study, we present novel data indicating that Dnr1 impacts on Dronc-dependent regulation of the apoptotic program. We show that depletion of Dnr1 results in elevated Dronc protein levels, which translates to increased caspase activation and activity upon induction of apoptosis. Conversely, we demonstrate that overexpression of Dnr1 blocks apoptotic caspase activity and prevents induction of apoptosis in tissue culture assays. Furthermore, we show that Dnr1 overexpression significantly reduces Dronc protein levels and identify the domains of Dnr1 necessary for these effects. From these data, we propose that Dnr1 inhibits initiator caspases in S2 cells.
Insights
Defense repressor 1 (Dnr1) regulates apoptosis by controlling initiator caspase levels. Depleting Dnr1 increases caspase activity, while overexpressing Dnr1 reduces it, suggesting Dnr1 inhibits caspases in Drosophila.
Area of Science:
- Cellular biology
- Molecular mechanisms of apoptosis
- Innate immunity signaling pathways
Background:
- Caspases are key regulators of apoptosis and NF-kappaB signaling.
- Inhibitor of apoptosis (IAP) proteins modulate caspase activity.
- Defense repressor 1 (Dnr1) is a RING-domain protein previously linked to NF-kappaB regulation.
Purpose of the Study:
- To investigate the role of Defense repressor 1 (Dnr1) in apoptosis regulation.
- To determine the impact of Dnr1 on Dronc-dependent apoptotic pathways.
- To elucidate the mechanism by which Dnr1 affects initiator caspase activity.
Main Methods:
- RNA interference (RNAi) for Dnr1 depletion in Drosophila S2 cells.
- Western blotting to assess Dronc protein levels.
- Caspase activity assays to measure apoptosis induction.
- Dnr1 domain analysis through overexpression studies.
Main Results:
- Depletion of Dnr1 led to increased Dronc protein levels, enhanced caspase activation, and heightened apoptotic activity.
- Overexpression of Dnr1 suppressed caspase activity and inhibited apoptosis induction.
- Dnr1 overexpression reduced Dronc protein levels, indicating a post-transcriptional regulatory mechanism.
Conclusions:
- Defense repressor 1 (Dnr1) plays a significant role in regulating the apoptotic program in Drosophila.
- Dnr1 acts as an inhibitor of initiator caspases, specifically impacting Dronc levels and activity.
- These findings reveal a novel function for Dnr1 in controlling apoptosis through modulation of caspase protein stability.
Related Concept Videos
01:17The Extrinsic Apoptotic Pathway
01:31The Intrinsic Apoptotic Pathway