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Updated: Jun 8, 2026
In Vitro Cleavage Assays using Purified Recombinant Drosophila Caspases for Substrate Screening
Published on: October 6, 2022
Drosophila IAP antagonists form multimeric complexes to promote cell death
Cristinel Sandu1, Hyung Don Ryoo, Hermann Steller
1Howard Hughes Medical Institute, Strang Laboratory of Apoptosis and Cancer Biology, The Rockefeller University, New York, NY 10065, USA.
Abstract:
Apoptosis is a specific form of cell death that is important for normal development and tissue homeostasis. Caspases are critical executioners of apoptosis, and living cells prevent their inappropriate activation through inhibitor of apoptosis proteins (IAPs). In Drosophila, caspase activation depends on the IAP antagonists, Reaper (Rpr), Head involution defective (Hid), and Grim. These proteins share a common motif to bind Drosophila IAP1 (DIAP1) and have partially redundant functions. We now show that IAP antagonists physically interact with each other. Rpr is able to self-associate and also binds to Hid and Grim. We have defined the domain involved in self-association and demonstrate that it is critical for cell-killing activity in vivo. In addition, we show that Rpr requires Hid for recruitment to the mitochondrial membrane and for efficient induction of cell death in vivo. Both targeting of Rpr to mitochondria and forced dimerization strongly promotes apoptosis. Our results reveal the functional importance of a previously unrecognized multimeric IAP antagonist complex for the induction of apoptosis.
Insights
Inhibitor of apoptosis antagonists, Reaper, Hid, and Grim, physically interact to form a complex. This multimeric complex is critical for inducing apoptosis, a key process in cell death.
Area of Science:
- Cell Biology
- Developmental Biology
- Molecular Biology
Background:
- Apoptosis is programmed cell death essential for development and homeostasis.
- Caspases execute apoptosis, but their activation is tightly regulated by inhibitor of apoptosis proteins (IAPs).
- In Drosophila, IAP antagonists like Reaper (Rpr), Head involution defective (Hid), and Grim are crucial for caspase activation.
Purpose of the Study:
- To investigate the physical interactions between Drosophila IAP antagonists.
- To determine the role of these interactions in the induction of apoptosis.
- To elucidate the mechanism by which IAP antagonists trigger cell death.
Main Methods:
- Co-immunoprecipitation assays to detect protein-protein interactions.
- Site-directed mutagenesis to identify functional domains.
- In vivo cell-killing assays in Drosophila.
- Mitochondrial localization studies.
Main Results:
- Reaper (Rpr), Head involution defective (Hid), and Grim physically interact, forming a multimeric complex.
- Rpr self-associates via a specific domain essential for its cell-killing activity.
- Rpr requires Hid for mitochondrial recruitment and efficient apoptosis induction.
- Forced dimerization and mitochondrial targeting of Rpr enhance apoptosis.
Conclusions:
- Drosophila IAP antagonists form a functional multimeric complex crucial for apoptosis induction.
- The interaction and self-association of IAP antagonists are key regulatory steps in the apoptotic pathway.
- This study reveals a previously unrecognized complex that controls programmed cell death.
Related Concept Videos
01:31The Intrinsic Apoptotic Pathway
01:17The Extrinsic Apoptotic Pathway