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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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DIAP1 auto-ubiquitination and interaction with Rpr and Hid. (A) SDS-PAGE gel showing E1 ubiquitin-activating enzyme Uba1 (Uba1-GST), E2 ubiquitin-conjugating enzyme UbcD1 (6His-UbcD1), 6His-ubiquitin (Ub), E3 ubiquitin ligase DIAP1 (6His-Flag-DIAP1), Rpr-His6, and HidΔMTS-His6, used in ubiquitination assays. Purification tags are not shown in the figure labeling. (B) In vitro coupling of Ub on UbcD1 (E2) in the absence (lane 1) or presence of Mg2+-ATP (lane 2). UbcD1-Ub adduct was detected by Coomassie staining. (C) In vitro DIAP1 auto-ubiquitination. Ubiquitination reactions containing E1, E2, Ub, and Flag-DIAP1, in the absence of Mg2+-ATP (lane 1) or in the presence of Mg2+-ATP (lane 2). The reaction was supplemented additionally with Rpr (lane 3), HidΔMTS (lane 4), or both (lane 5). Flag-DIAP1 was immunoprecipitated with anti-FLAG resin. Polyubiquitination species were detected in Western blot with an anti-ubiquitin antibody. (D) Coomassie-stained SDS-PAGE gel, showing the coimmunoprecipitation of Flag-DIAP1 with Rpr and HidΔMTS. “Input” shows the amount of Flag-DIAP1 (lane 1), Rpr (lane 2), or HidΔMTS (lane 3) used for co-immunoprecipitation. “IP:Flag” shows the anti-FLAG coimmunoprecipitation fractions. Lane 4 indicates the amount of Flag-DIAP1 recovered by the anti-FLAG resin. Lane 5 shows the coimmunoprecipitation of Rpr with Flag-DIAP1. Lane 6 shows the coimmunoprecipitation of HidΔMTS with Flag-DIAP1. Lane 7 shows the coimmunoprecipitation of HidΔMTS and Rpr with Flag-DIAP1.
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fig6: DIAP1 auto-ubiquitination and interaction with Rpr and Hid. (A) SDS-PAGE gel showing E1 ubiquitin-activating enzyme Uba1 (Uba1-GST), E2 ubiquitin-conjugating enzyme UbcD1 (6His-UbcD1), 6His-ubiquitin (Ub), E3 ubiquitin ligase DIAP1 (6His-Flag-DIAP1), Rpr-His6, and HidΔMTS-His6, used in ubiquitination assays. Purification tags are not shown in the figure labeling. (B) In vitro coupling of Ub on UbcD1 (E2) in the absence (lane 1) or presence of Mg2+-ATP (lane 2). UbcD1-Ub adduct was detected by Coomassie staining. (C) In vitro DIAP1 auto-ubiquitination. Ubiquitination reactions containing E1, E2, Ub, and Flag-DIAP1, in the absence of Mg2+-ATP (lane 1) or in the presence of Mg2+-ATP (lane 2). The reaction was supplemented additionally with Rpr (lane 3), HidΔMTS (lane 4), or both (lane 5). Flag-DIAP1 was immunoprecipitated with anti-FLAG resin. Polyubiquitination species were detected in Western blot with an anti-ubiquitin antibody. (D) Coomassie-stained SDS-PAGE gel, showing the coimmunoprecipitation of Flag-DIAP1 with Rpr and HidΔMTS. “Input” shows the amount of Flag-DIAP1 (lane 1), Rpr (lane 2), or HidΔMTS (lane 3) used for co-immunoprecipitation. “IP:Flag” shows the anti-FLAG coimmunoprecipitation fractions. Lane 4 indicates the amount of Flag-DIAP1 recovered by the anti-FLAG resin. Lane 5 shows the coimmunoprecipitation of Rpr with Flag-DIAP1. Lane 6 shows the coimmunoprecipitation of HidΔMTS with Flag-DIAP1. Lane 7 shows the coimmunoprecipitation of HidΔMTS and Rpr with Flag-DIAP1.

Mentions: Why does Rpr kill better in the presence of Hid? To answer this question we started from the premise that both IAP antagonists act to stimulate DIAP1 auto-ubiquitination (Hays et al., 2002; Ryoo et al., 2002; Yoo et al., 2002). To this end, we purified all components involved in DIAP1 auto-ubiquitination, namely Uba1 (E1), UbcD1 (E2), ubiquitin (Ub), DIAP1, Rpr, and HidΔMTS from E. coli (Fig. 6 A). HidΔMTS entails residues 1–386. The last 24 amino acids (387–410) of Hid, which constitute the membrane-inserted mitochondrial targeting sequence, were deleted to produce protein soluble for biochemical assays. When designing this construct, we inspected Hid secondary structure to avoid terminating the protein inside a secondary structure element (unpublished data). The proteins are active, as we could reconstitute the covalent coupling of one Ub molecule on UbcD1-conjugating enzyme, in an Uba1- and Mg2+-ATP–dependent fashion (Fig. 6 B). Furthermore, by using reducing agents to break down the E2-Ub thiolesters, we confirmed the presence of the UbcD1-Ub adduct (unpublished data). We next examined DIAP1 ubiquitination in the presence of Mg2+-ATP, Rpr, and/or HidΔMTS. Although DIAP1 does not self-ubiquitinate in the presence of Mg2+-ATP (Fig. 6 C), a dramatic transfer of ubiquitin to DIAP1 could be observed when the reaction is supplemented with Rpr. Thus, we have fully reconstituted in vitro a DIAP1 auto-ubiquitinating complex from Drosophila. Because IAP antagonists bind DIAP1 with conserved motifs it is often assumed that the mechanism of DIAP1 inactivation should also be conserved. However, when HidΔMTS was added in the DIAP1 ubiquitination assay instead of Rpr, no DIAP1 auto-ubiquitination could be observed. Despite good solubility, HidΔMTS did not stimulate DIAP1 degradation. When HidΔMTS and Rpr were added together to the DIAP1 ubiquitination assay, HidΔMTS did not enhance Rpr-dependent DIAP1 ubiquitination. The inability of HidΔMTS to induce DIAP1 ubiquitination could be a result of the following reasons. First, it is possible that recombinant HidΔMTS does not reflect endogenous Hid function, perhaps due to the deletion of its C-terminal hydrophobic region. A second possibility is that Rpr and Hid have different mechanisms of DIAP1 inactivation and only Rpr induces DIAP1 ubiquitination, Hid having a different role in DIAP1 inactivation. In an attempt to address these possibilities, we examined the interactions between DIAP1, Rpr, and Hid using the purified proteins used in the ubiquitination assay. Under these conditions, HidΔMTS was able to bind DIAP1 at a roughly equimolar ratio as judged by band intensity on SDS-PAGE gel, despite HidΔMTS’s inability to stimulate DIAP1 ubiquitination (Fig. 6 D). Furthermore, we have examined the ability of HidΔMTS to form oligomers by formaldehyde cross-linking experiments. Purified HidΔMTS and Rpr appear to form oligomers under these conditions (Fig. S3 B). In addition, the interaction between Rpr and HidΔMTS was already shown in Fig. 3 B. These experiments indicate that deletion of Hid’s MTS does not block its ability to oligomerize or interact with Rpr and DIAP1. We have next examined the ability of full-length Hid or HidΔMTS to induce DIAP1ΔR degradation in HEK293 cells. Unlike Rpr (Fig. 2 F), full-length Hid and HidΔMTS do not induce DIAP1ΔR degradation (Fig. S3 C). These observations and others suggest that most probably Hid does not induce DIAP1 ubiquitination directly.


Drosophila IAP antagonists form multimeric complexes to promote cell death.

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

DIAP1 auto-ubiquitination and interaction with Rpr and Hid. (A) SDS-PAGE gel showing E1 ubiquitin-activating enzyme Uba1 (Uba1-GST), E2 ubiquitin-conjugating enzyme UbcD1 (6His-UbcD1), 6His-ubiquitin (Ub), E3 ubiquitin ligase DIAP1 (6His-Flag-DIAP1), Rpr-His6, and HidΔMTS-His6, used in ubiquitination assays. Purification tags are not shown in the figure labeling. (B) In vitro coupling of Ub on UbcD1 (E2) in the absence (lane 1) or presence of Mg2+-ATP (lane 2). UbcD1-Ub adduct was detected by Coomassie staining. (C) In vitro DIAP1 auto-ubiquitination. Ubiquitination reactions containing E1, E2, Ub, and Flag-DIAP1, in the absence of Mg2+-ATP (lane 1) or in the presence of Mg2+-ATP (lane 2). The reaction was supplemented additionally with Rpr (lane 3), HidΔMTS (lane 4), or both (lane 5). Flag-DIAP1 was immunoprecipitated with anti-FLAG resin. Polyubiquitination species were detected in Western blot with an anti-ubiquitin antibody. (D) Coomassie-stained SDS-PAGE gel, showing the coimmunoprecipitation of Flag-DIAP1 with Rpr and HidΔMTS. “Input” shows the amount of Flag-DIAP1 (lane 1), Rpr (lane 2), or HidΔMTS (lane 3) used for co-immunoprecipitation. “IP:Flag” shows the anti-FLAG coimmunoprecipitation fractions. Lane 4 indicates the amount of Flag-DIAP1 recovered by the anti-FLAG resin. Lane 5 shows the coimmunoprecipitation of Rpr with Flag-DIAP1. Lane 6 shows the coimmunoprecipitation of HidΔMTS with Flag-DIAP1. Lane 7 shows the coimmunoprecipitation of HidΔMTS and Rpr with Flag-DIAP1.
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fig6: DIAP1 auto-ubiquitination and interaction with Rpr and Hid. (A) SDS-PAGE gel showing E1 ubiquitin-activating enzyme Uba1 (Uba1-GST), E2 ubiquitin-conjugating enzyme UbcD1 (6His-UbcD1), 6His-ubiquitin (Ub), E3 ubiquitin ligase DIAP1 (6His-Flag-DIAP1), Rpr-His6, and HidΔMTS-His6, used in ubiquitination assays. Purification tags are not shown in the figure labeling. (B) In vitro coupling of Ub on UbcD1 (E2) in the absence (lane 1) or presence of Mg2+-ATP (lane 2). UbcD1-Ub adduct was detected by Coomassie staining. (C) In vitro DIAP1 auto-ubiquitination. Ubiquitination reactions containing E1, E2, Ub, and Flag-DIAP1, in the absence of Mg2+-ATP (lane 1) or in the presence of Mg2+-ATP (lane 2). The reaction was supplemented additionally with Rpr (lane 3), HidΔMTS (lane 4), or both (lane 5). Flag-DIAP1 was immunoprecipitated with anti-FLAG resin. Polyubiquitination species were detected in Western blot with an anti-ubiquitin antibody. (D) Coomassie-stained SDS-PAGE gel, showing the coimmunoprecipitation of Flag-DIAP1 with Rpr and HidΔMTS. “Input” shows the amount of Flag-DIAP1 (lane 1), Rpr (lane 2), or HidΔMTS (lane 3) used for co-immunoprecipitation. “IP:Flag” shows the anti-FLAG coimmunoprecipitation fractions. Lane 4 indicates the amount of Flag-DIAP1 recovered by the anti-FLAG resin. Lane 5 shows the coimmunoprecipitation of Rpr with Flag-DIAP1. Lane 6 shows the coimmunoprecipitation of HidΔMTS with Flag-DIAP1. Lane 7 shows the coimmunoprecipitation of HidΔMTS and Rpr with Flag-DIAP1.
Mentions: Why does Rpr kill better in the presence of Hid? To answer this question we started from the premise that both IAP antagonists act to stimulate DIAP1 auto-ubiquitination (Hays et al., 2002; Ryoo et al., 2002; Yoo et al., 2002). To this end, we purified all components involved in DIAP1 auto-ubiquitination, namely Uba1 (E1), UbcD1 (E2), ubiquitin (Ub), DIAP1, Rpr, and HidΔMTS from E. coli (Fig. 6 A). HidΔMTS entails residues 1–386. The last 24 amino acids (387–410) of Hid, which constitute the membrane-inserted mitochondrial targeting sequence, were deleted to produce protein soluble for biochemical assays. When designing this construct, we inspected Hid secondary structure to avoid terminating the protein inside a secondary structure element (unpublished data). The proteins are active, as we could reconstitute the covalent coupling of one Ub molecule on UbcD1-conjugating enzyme, in an Uba1- and Mg2+-ATP–dependent fashion (Fig. 6 B). Furthermore, by using reducing agents to break down the E2-Ub thiolesters, we confirmed the presence of the UbcD1-Ub adduct (unpublished data). We next examined DIAP1 ubiquitination in the presence of Mg2+-ATP, Rpr, and/or HidΔMTS. Although DIAP1 does not self-ubiquitinate in the presence of Mg2+-ATP (Fig. 6 C), a dramatic transfer of ubiquitin to DIAP1 could be observed when the reaction is supplemented with Rpr. Thus, we have fully reconstituted in vitro a DIAP1 auto-ubiquitinating complex from Drosophila. Because IAP antagonists bind DIAP1 with conserved motifs it is often assumed that the mechanism of DIAP1 inactivation should also be conserved. However, when HidΔMTS was added in the DIAP1 ubiquitination assay instead of Rpr, no DIAP1 auto-ubiquitination could be observed. Despite good solubility, HidΔMTS did not stimulate DIAP1 degradation. When HidΔMTS and Rpr were added together to the DIAP1 ubiquitination assay, HidΔMTS did not enhance Rpr-dependent DIAP1 ubiquitination. The inability of HidΔMTS to induce DIAP1 ubiquitination could be a result of the following reasons. First, it is possible that recombinant HidΔMTS does not reflect endogenous Hid function, perhaps due to the deletion of its C-terminal hydrophobic region. A second possibility is that Rpr and Hid have different mechanisms of DIAP1 inactivation and only Rpr induces DIAP1 ubiquitination, Hid having a different role in DIAP1 inactivation. In an attempt to address these possibilities, we examined the interactions between DIAP1, Rpr, and Hid using the purified proteins used in the ubiquitination assay. Under these conditions, HidΔMTS was able to bind DIAP1 at a roughly equimolar ratio as judged by band intensity on SDS-PAGE gel, despite HidΔMTS’s inability to stimulate DIAP1 ubiquitination (Fig. 6 D). Furthermore, we have examined the ability of HidΔMTS to form oligomers by formaldehyde cross-linking experiments. Purified HidΔMTS and Rpr appear to form oligomers under these conditions (Fig. S3 B). In addition, the interaction between Rpr and HidΔMTS was already shown in Fig. 3 B. These experiments indicate that deletion of Hid’s MTS does not block its ability to oligomerize or interact with Rpr and DIAP1. We have next examined the ability of full-length Hid or HidΔMTS to induce DIAP1ΔR degradation in HEK293 cells. Unlike Rpr (Fig. 2 F), full-length Hid and HidΔMTS do not induce DIAP1ΔR degradation (Fig. S3 C). These observations and others suggest that most probably Hid does not induce DIAP1 ubiquitination directly.

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