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Drosophila IAP antagonists form multimeric complexes to promote cell death.

Sandu C, Ryoo HD, Steller H - J. Cell Biol. (2010)

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Howard 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.

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Enforced Rpr dimers kill by apoptosis in Drosophila. (A) Amino acid sequences and structural elements of Rpr dimers. RprLZ is an enforced parallel Rpr dimer where Rpr helical region (residues 10–46) was replaced with a parallel leucine zipper (GCN4), whereas RprProP is an enforced anti-parallel Rpr-dimer. LZ and ProP amino acid sequences are represented in blue. Residues in brown were inserted on both sides of each dimerization domain to preserve the same length as wild-type Rpr. IBMRpr and TailRpr are identical as in wild-type Rpr. A secondary structure prediction is represented below each sequence. Nomenclature: c, disordered; e, β-strand; h, helical. All constructs have attached a C-terminal HA tag, not represented in this diagram. To the right are schematic representations of RprLZ and RprProP with the IBMRpr (shown in red), ribbon representations of the dimerization domains LZ (PDB #2ZTA) and ProP (PDB #1R48) (shown in blue) and the TailRpr (shown in black). Note the position of the IBM motifs in RprLZ and RprProP. (B) Drosophila eye images from transgenic flies expressing RprLZ-HA or RprProP-HA. Genotypes: ;UAS:RprLZ-HA/GMR>Gal4; and ;UAS:RprProP-HA/GMR>Gal4;. (C) Eye-antennal imaginal discs from third instar transgenic larvae, expressing RprLZ-HA and RprProP-HA, stained with an anti-HA antibody. Genotype: UAS:p35/+;UAS:RprLZ-HA/GMR>Gal4; and UAS:p35/+;UAS:RprProP-HA/GMR>Gal4;. (D) Rescue of the RprLZ-HA induced eye ablation by Rpr-insensitive diap1 alleles or p35. Genotypes: ;UAS:RprLZ-HA/GMR>Gal4;, ;UAS:RprLZ-HA/GMR>Gal4;diap16-3s/+, ;UAS:RprLZ-HA/GMR>Gal4;diap123-4s/+ and UAS:p35/+;UAS:RprLZ-HA/GMR>Gal4;. (E) Rescue of the Rpr-HA induced eye ablation by Rpr-insensitive diap1 alleles or p35. Genotypes are identical to D, except that UAS:RprLZ-HA was replaced with UAS:Rpr-HA. (F) Ectopic expression of DIAP1ΔR-Flag or coexpression with Rpr-HA or RprLZ-HA in HEK293 cells, showing the ability of Rpr and RprLZ to induce DIAP1 degradation. Actin was used as a loading control. (G) Overexpression of RprLZ-HA in the presence of p35 in the posterior compartment of the wing discs and its effect on DIAP1 level. Expression of RprLZ was detected with an anti-HA antibody, whereas DIAP1 was immunostained with a rabbit anti-DIAP1 antibody.
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fig2: Enforced Rpr dimers kill by apoptosis in Drosophila. (A) Amino acid sequences and structural elements of Rpr dimers. RprLZ is an enforced parallel Rpr dimer where Rpr helical region (residues 10–46) was replaced with a parallel leucine zipper (GCN4), whereas RprProP is an enforced anti-parallel Rpr-dimer. LZ and ProP amino acid sequences are represented in blue. Residues in brown were inserted on both sides of each dimerization domain to preserve the same length as wild-type Rpr. IBMRpr and TailRpr are identical as in wild-type Rpr. A secondary structure prediction is represented below each sequence. Nomenclature: c, disordered; e, β-strand; h, helical. All constructs have attached a C-terminal HA tag, not represented in this diagram. To the right are schematic representations of RprLZ and RprProP with the IBMRpr (shown in red), ribbon representations of the dimerization domains LZ (PDB #2ZTA) and ProP (PDB #1R48) (shown in blue) and the TailRpr (shown in black). Note the position of the IBM motifs in RprLZ and RprProP. (B) Drosophila eye images from transgenic flies expressing RprLZ-HA or RprProP-HA. Genotypes: ;UAS:RprLZ-HA/GMR>Gal4; and ;UAS:RprProP-HA/GMR>Gal4;. (C) Eye-antennal imaginal discs from third instar transgenic larvae, expressing RprLZ-HA and RprProP-HA, stained with an anti-HA antibody. Genotype: UAS:p35/+;UAS:RprLZ-HA/GMR>Gal4; and UAS:p35/+;UAS:RprProP-HA/GMR>Gal4;. (D) Rescue of the RprLZ-HA induced eye ablation by Rpr-insensitive diap1 alleles or p35. Genotypes: ;UAS:RprLZ-HA/GMR>Gal4;, ;UAS:RprLZ-HA/GMR>Gal4;diap16-3s/+, ;UAS:RprLZ-HA/GMR>Gal4;diap123-4s/+ and UAS:p35/+;UAS:RprLZ-HA/GMR>Gal4;. (E) Rescue of the Rpr-HA induced eye ablation by Rpr-insensitive diap1 alleles or p35. Genotypes are identical to D, except that UAS:RprLZ-HA was replaced with UAS:Rpr-HA. (F) Ectopic expression of DIAP1ΔR-Flag or coexpression with Rpr-HA or RprLZ-HA in HEK293 cells, showing the ability of Rpr and RprLZ to induce DIAP1 degradation. Actin was used as a loading control. (G) Overexpression of RprLZ-HA in the presence of p35 in the posterior compartment of the wing discs and its effect on DIAP1 level. Expression of RprLZ was detected with an anti-HA antibody, whereas DIAP1 was immunostained with a rabbit anti-DIAP1 antibody.

Mentions: Although the above experiments indicate that Rpr self-association is required for its pro-apoptotic activity, whether it is sufficient to recapitulate Rpr’s pro-apoptotic function remained unclear. To test this, we replaced the helical domain of Rpr (residues 10–46) with well-defined dimerization domains from heterologous proteins whose three-dimensional structures have been previously determined. Specifically, we used a parallel leucine zipper (LZ) from the yeast transcription factor GCN4 (O’Shea et al., 1991) and an anti-parallel coiled-coil domain from the Escherichia coli osmosensor ProP (Zoetewey et al., 2003; Fig. 2 A). When these chimeric proteins were expressed in the fly eye using the GMR>Gal4/UAS system we found that RprLZ triggered massive cell death, as evidenced by the partially ablated eye structure (Fig. 2 B), supporting the idea that Rpr dimerization is sufficient to account for its central helical domain’s function. On the other hand, RprProP did not trigger cell death under similar conditions, despite being expressed at similar levels with RprLZ (Fig. 2 C). Next, we examined whether RprLZ induces cell death through a mechanism similar to the wild-type Rpr, namely the inhibition of DIAP1 and activation of caspases. Supporting the requirement of caspase activation, coexpression of p35, a well-established caspase inhibitor of viral origin, rescued the eye morphology caused by RprLZ (Fig. 2 D, right) as well as wild-type Rpr (Fig. 2 E, right). To test the requirement of DIAP1 inactivation, we took advantage of the diap16-3s and diap123-4s alleles, which are endogenous alleles bearing point mutations in the IBM-binding pocket of DIAP1 BIR domains, making cells resistant to Rpr-induced cell death (Goyal et al., 2000). The presence of these diap1 alleles in the background significantly suppressed apoptosis induced by the RprLZ (Fig. 2 D) as well as wild-type Rpr (Fig. 2 E). Next, we examined the ability of RprLZ to induce DIAP1 degradation. In coexpression experiments in HEK293 cells, RprLZ was able to stimulate DIAP1ΔR degradation to significant extent (Fig. 2 F, right), but lower than wild-type Rpr (Fig. 2 F, left). DIAP1ΔR was used instead of full-length DIAP1 due to its increased stability (not depicted). Ability of RprLZ to induce DIAP1 degradation was also shown in wing discs, after overexpression in the presence of p35 (Fig. 2 G). These results support the idea that RprLZ has pro-apoptotic mechanisms similar to that of wild-type Rpr.


Drosophila IAP antagonists form multimeric complexes to promote cell death.

Sandu C, Ryoo HD, Steller H - J. Cell Biol. (2010)

Enforced Rpr dimers kill by apoptosis in Drosophila. (A) Amino acid sequences and structural elements of Rpr dimers. RprLZ is an enforced parallel Rpr dimer where Rpr helical region (residues 10–46) was replaced with a parallel leucine zipper (GCN4), whereas RprProP is an enforced anti-parallel Rpr-dimer. LZ and ProP amino acid sequences are represented in blue. Residues in brown were inserted on both sides of each dimerization domain to preserve the same length as wild-type Rpr. IBMRpr and TailRpr are identical as in wild-type Rpr. A secondary structure prediction is represented below each sequence. Nomenclature: c, disordered; e, β-strand; h, helical. All constructs have attached a C-terminal HA tag, not represented in this diagram. To the right are schematic representations of RprLZ and RprProP with the IBMRpr (shown in red), ribbon representations of the dimerization domains LZ (PDB #2ZTA) and ProP (PDB #1R48) (shown in blue) and the TailRpr (shown in black). Note the position of the IBM motifs in RprLZ and RprProP. (B) Drosophila eye images from transgenic flies expressing RprLZ-HA or RprProP-HA. Genotypes: ;UAS:RprLZ-HA/GMR>Gal4; and ;UAS:RprProP-HA/GMR>Gal4;. (C) Eye-antennal imaginal discs from third instar transgenic larvae, expressing RprLZ-HA and RprProP-HA, stained with an anti-HA antibody. Genotype: UAS:p35/+;UAS:RprLZ-HA/GMR>Gal4; and UAS:p35/+;UAS:RprProP-HA/GMR>Gal4;. (D) Rescue of the RprLZ-HA induced eye ablation by Rpr-insensitive diap1 alleles or p35. Genotypes: ;UAS:RprLZ-HA/GMR>Gal4;, ;UAS:RprLZ-HA/GMR>Gal4;diap16-3s/+, ;UAS:RprLZ-HA/GMR>Gal4;diap123-4s/+ and UAS:p35/+;UAS:RprLZ-HA/GMR>Gal4;. (E) Rescue of the Rpr-HA induced eye ablation by Rpr-insensitive diap1 alleles or p35. Genotypes are identical to D, except that UAS:RprLZ-HA was replaced with UAS:Rpr-HA. (F) Ectopic expression of DIAP1ΔR-Flag or coexpression with Rpr-HA or RprLZ-HA in HEK293 cells, showing the ability of Rpr and RprLZ to induce DIAP1 degradation. Actin was used as a loading control. (G) Overexpression of RprLZ-HA in the presence of p35 in the posterior compartment of the wing discs and its effect on DIAP1 level. Expression of RprLZ was detected with an anti-HA antibody, whereas DIAP1 was immunostained with a rabbit anti-DIAP1 antibody.
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fig2: Enforced Rpr dimers kill by apoptosis in Drosophila. (A) Amino acid sequences and structural elements of Rpr dimers. RprLZ is an enforced parallel Rpr dimer where Rpr helical region (residues 10–46) was replaced with a parallel leucine zipper (GCN4), whereas RprProP is an enforced anti-parallel Rpr-dimer. LZ and ProP amino acid sequences are represented in blue. Residues in brown were inserted on both sides of each dimerization domain to preserve the same length as wild-type Rpr. IBMRpr and TailRpr are identical as in wild-type Rpr. A secondary structure prediction is represented below each sequence. Nomenclature: c, disordered; e, β-strand; h, helical. All constructs have attached a C-terminal HA tag, not represented in this diagram. To the right are schematic representations of RprLZ and RprProP with the IBMRpr (shown in red), ribbon representations of the dimerization domains LZ (PDB #2ZTA) and ProP (PDB #1R48) (shown in blue) and the TailRpr (shown in black). Note the position of the IBM motifs in RprLZ and RprProP. (B) Drosophila eye images from transgenic flies expressing RprLZ-HA or RprProP-HA. Genotypes: ;UAS:RprLZ-HA/GMR>Gal4; and ;UAS:RprProP-HA/GMR>Gal4;. (C) Eye-antennal imaginal discs from third instar transgenic larvae, expressing RprLZ-HA and RprProP-HA, stained with an anti-HA antibody. Genotype: UAS:p35/+;UAS:RprLZ-HA/GMR>Gal4; and UAS:p35/+;UAS:RprProP-HA/GMR>Gal4;. (D) Rescue of the RprLZ-HA induced eye ablation by Rpr-insensitive diap1 alleles or p35. Genotypes: ;UAS:RprLZ-HA/GMR>Gal4;, ;UAS:RprLZ-HA/GMR>Gal4;diap16-3s/+, ;UAS:RprLZ-HA/GMR>Gal4;diap123-4s/+ and UAS:p35/+;UAS:RprLZ-HA/GMR>Gal4;. (E) Rescue of the Rpr-HA induced eye ablation by Rpr-insensitive diap1 alleles or p35. Genotypes are identical to D, except that UAS:RprLZ-HA was replaced with UAS:Rpr-HA. (F) Ectopic expression of DIAP1ΔR-Flag or coexpression with Rpr-HA or RprLZ-HA in HEK293 cells, showing the ability of Rpr and RprLZ to induce DIAP1 degradation. Actin was used as a loading control. (G) Overexpression of RprLZ-HA in the presence of p35 in the posterior compartment of the wing discs and its effect on DIAP1 level. Expression of RprLZ was detected with an anti-HA antibody, whereas DIAP1 was immunostained with a rabbit anti-DIAP1 antibody.
Mentions: Although the above experiments indicate that Rpr self-association is required for its pro-apoptotic activity, whether it is sufficient to recapitulate Rpr’s pro-apoptotic function remained unclear. To test this, we replaced the helical domain of Rpr (residues 10–46) with well-defined dimerization domains from heterologous proteins whose three-dimensional structures have been previously determined. Specifically, we used a parallel leucine zipper (LZ) from the yeast transcription factor GCN4 (O’Shea et al., 1991) and an anti-parallel coiled-coil domain from the Escherichia coli osmosensor ProP (Zoetewey et al., 2003; Fig. 2 A). When these chimeric proteins were expressed in the fly eye using the GMR>Gal4/UAS system we found that RprLZ triggered massive cell death, as evidenced by the partially ablated eye structure (Fig. 2 B), supporting the idea that Rpr dimerization is sufficient to account for its central helical domain’s function. On the other hand, RprProP did not trigger cell death under similar conditions, despite being expressed at similar levels with RprLZ (Fig. 2 C). Next, we examined whether RprLZ induces cell death through a mechanism similar to the wild-type Rpr, namely the inhibition of DIAP1 and activation of caspases. Supporting the requirement of caspase activation, coexpression of p35, a well-established caspase inhibitor of viral origin, rescued the eye morphology caused by RprLZ (Fig. 2 D, right) as well as wild-type Rpr (Fig. 2 E, right). To test the requirement of DIAP1 inactivation, we took advantage of the diap16-3s and diap123-4s alleles, which are endogenous alleles bearing point mutations in the IBM-binding pocket of DIAP1 BIR domains, making cells resistant to Rpr-induced cell death (Goyal et al., 2000). The presence of these diap1 alleles in the background significantly suppressed apoptosis induced by the RprLZ (Fig. 2 D) as well as wild-type Rpr (Fig. 2 E). Next, we examined the ability of RprLZ to induce DIAP1 degradation. In coexpression experiments in HEK293 cells, RprLZ was able to stimulate DIAP1ΔR degradation to significant extent (Fig. 2 F, right), but lower than wild-type Rpr (Fig. 2 F, left). DIAP1ΔR was used instead of full-length DIAP1 due to its increased stability (not depicted). Ability of RprLZ to induce DIAP1 degradation was also shown in wing discs, after overexpression in the presence of p35 (Fig. 2 G). These results support the idea that RprLZ has pro-apoptotic mechanisms similar to that of wild-type Rpr.

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Howard 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.

Show MeSH
Related in: MedlinePlus