Dcp-1 overexpression induces apoptosis independent of the apoptosome.

(A) Schematic structures and intrinsically disordered region prediction of Dcp-1 (dark cyan) and Drice (red). The predicted disordered score plot is shown. Regions wherein the score is more than 0.5 are predicted to be disordered. (B) Representative images of female wing phenotypes upon caspase overexpression using WP-Gal4. (C) Representative images of larval wing imaginal disc upon 3xHA-tagged executioner caspase (magenta) overexpression using WP-Gal4. Nuclei are visualized by Hoechst 33342 (green). Magnified images are shown in right below. Arrowheads indicate condensed nuclei. Scale bar: 50 µm (top) and 10 µm (bottom). (D) Schematic diagram of canonical apoptosis signaling in Drosophila. (E, F) Representative images of female wing phenotypes upon Dcp-1::VENUS overexpression using WP-Gal4 with the genetic perturbation of canonical apoptosis signaling. (G) Quantification of (E) and (F). Each wing is manually classified into four (wingless, severe, mild, and intact) categories. Schematic images of the four categories are shown on the right. Sample sizes are shown in the figure.

Dcp-1-enriched proximal proteins are required for Dcp-1 activation.

(A) Western blotting of expression of C-terminally V5::TurboID knocked-in tagged caspases and biotinylation patterns of their proximal proteins detected by streptavidin (SA) in larval wing imaginal discs. (B) A schematic diagram of the TurboID-mediated identification of proximal proteins. Dcp-1- and Drice-proximal proteins (Protein C, D, E, F, G) are promiscuously labeled in vivo with the administration of biotin. Then, biotinylated proteins are purified using NeutrAvidin magnetic beads and are subsequently analyzed by mass spectrometry. (C) A summary of mass spectrometry analysis of proteins in the larval imaginal discs. Among 818 proteins identified, 230 proteins were detected as highly enriched proteins in Drice::V5::TurboID flies (FC [Drice::V5::TurboID/Dcp-1::V5::TurboID] > 2) and 16 proteins were detected as highly enriched proteins in Dcp-1::V5::TurboID flies flies (FC [Dcp-1::V5::TurboID/Drice::V5::TurboID] > 2). (D) Western blotting validation of the anti-CCT8 antibody in whole larval lysates without the digestive tract and fat body. The anti-CCT8 antibody detects both endogenous CCT8 (MW: 59.4 kDa) and 3xFLAG::StrepII::VENUS::StrepII (FSVS)-tagged CCT8 (MW: 91.1 kDa). (E) Western blotting of endogenous CCT8 expressed in larval wing imaginal disc. Dcp-1-proximal and Drice-proximal proteins biotinylated by C-terminally knocked-in tagged V5::TurboID are purified using NeutrAvidin. The red asterisk represents a non-specific band in the marker lane. (F) Schematic diagram of the RNAi screen targeting eight Dcp-1-enriched proximal proteins. The WP > Dcp-1::VENUS line was crossed with each UAS-RNAi line, and wing phenotypes of the F1 progeny were scored. (G) Representative images of female wing phenotypes from Dcp-1::VENUS overexpression driven by WP-Gal4 with knockdown of Dcp-1-enriched proximal proteins. “XX” denotes the knocked-down gene, and “#1” and “#2” indicate independent RNAi lines. (H) Representative images of female wing phenotypes upon Dcp-1::VENUS overexpression driven by WP-Gal4 in the Sirt1 null mutant background. (I) Quantification of (H). Each wing is manually classified into four (wingless, severe, mild, and intact) categories. Sample sizes are shown in the figure.

Debcl, Buffy, and autophagy are specifically required for Dcp-1 overexpression-induced apoptosis.

(A) Representative images of female wing phenotypes from Dcp-1::VENUS overexpression driven by WP-Gal4 with the genetic perturbation of Synr, Debcl, Buffy, and autophagy. “XX” denotes the knocked-down gene, and “#1” and “#2” indicate independent RNAi lines. (B) Quantification of (A). “XX” denotes the knocked-down gene, and “#1” and “#2” indicate independent RNAi lines. Each wing is manually classified into four (wingless, severe, mild, and intact) categories. Sample sizes are shown in the figure. (C) Representative images of female eye phenotypes upon Hid overexpression with the genetic perturbation of canonical apoptosis signaling. (D) Representative images of female eye phenotypes upon Hid overexpression with the genetic perturbation of Sirt1 or Fkbp59. “#1” and “#2” indicate independent RNAi lines. (E) Representative images of female eye phenotypes upon Hid overexpression with the genetic perturbation of Debcl, Buffy, and autophagy.

Cleaved Dcp-1, but not Drice, interacts with Bruce.

(A) Domain structure of Bruce. LD denotes the linker domain, WD40 a WD40 domain, BIR the Baculoviral IAP repeat, â-X a â-sandwich-like fold, CBM a domain resembling the carbohydrate-binding module family 32 (CBM), UBL the ubiquitin-like domain, and UBC the E2-E3 hybrid ubiquitin-conjugating (UBC) domain. The region used for structural prediction using AlphaFold3 is indicated in red. (B) IBM-like sequence in Dcp-1. The propeptide cleavage site of Dcp-1 is indicated by a black dashed line; the caspase recognition motif is highlighted in black; the IBM-like sequence is highlighted in pink; and the region used for AlphaFold3 prediction is shown in red. (C) IBM-like sequence in Drice. The propeptide cleavage site of Drice is indicated by a black dashed line; the caspase recognition motif is highlighted in black; the IBM-like sequence is highlighted in pink; and the region used for AlphaFold3 prediction is shown in red. (D) Predicted complex structure of Bruce BIR and Dcp-1 IBM-like sequence. The AlphaFold3 prediction [model 0] is shown. The Bruce β-X2 and BIR domains are displayed as surfaces in green and pink, respectively. Dcp-1 is shown in stick representation and color-coded according to pLDDT scores, as indicated in the figure. See also panel (F). (E) Predicted complex structure of Bruce BIR and Drice IBM-like sequence. The AlphaFold3 prediction [model 0] is shown. The Bruce â-X2 and BIR domains are displayed as surfaces in green and pink, respectively. Drice is shown in stick representation and color-coded according to pLDDT scores, as indicated in the figure. See also panel (G). (F) pLDDT scores of Dcp-1 in the five predicted complex structures with Bruce. For all five AlphaFold3 models, per-residue pLDDT scores of Dcp-1 are plotted. Prediction confidence is color-coded as follows: very high (pLDDT > 90) in dark blue, confident (90 > pLDDT > 70) in light blue, low (70 > pLDDT > 50) in yellow, and very low (pLDDT < 50) in orange. (G) pLDDT scores of Drice in the five predicted complex structures with dBruce. For all five AlphaFold3 models, per-residue pLDDT scores of Drice are plotted. Prediction confidence is color-coded as follows: very high (pLDDT > 90) in dark blue, confident (90 > pLDDT > 70) in light blue, low (70 > pLDDT > 50) in yellow, and very low (pLDDT < 50) in orange. (H) Co-immunoprecipitation of N-terminally 3xFLAG-tagged N-terminal region of Bruce (Br, 1–900 aa; containing LD, BIR, and WD40 domains) with C-terminally myc-tagged, catalytically inactive Dcp-1 or Drice (catalytic cysteine mutated to glycine; CG) expressed in Drosophila S2 cells. N-terminally 3xFLAG-tagged mRuby3 (mRb3) was used as a control. Apoptosis was induced by cycloheximide (CHX) treatment. 3xFLAG-tagged proteins were immunoprecipitated using an anti-3xFLAG antibody.

Bruce selectively suppresses Dcp-1 activity and wing tissue growth.

(A) Expression pattern of overexpressed myc::Bruce (green) by using WP-Gal4 in the wing imaginal discs. Nuclei are visualized using Hoechst 33342 (magenta). Scale bar: 50 µm. (B) Representative images of female wing phenotypes from Dcp-1::VENUS overexpression driven by WP-Gal4 with DIAP1 or Bruce overexpression. (C) Quantification of (B). Each wing is manually classified into four (wingless, severe, mild, and intact) categories. Sample sizes are shown in the figure. (D) Representative images of female wing phenotypes upon Synr overexpression driven by Nub-Gal4 with DIAP1 or Bruce overexpression. (E) Representative images of female eye phenotypes upon Hid overexpression with DIAP1 or Bruce overexpression. (F) Representative images of female wing upon Bruce or Bruce-RNAi (Bruce-i) overexpression using WP-Gal4. Scale bar: 1 mm. (G) Quantification of relative wing size normalized to the corresponding No-Gal4 control. Data are presented as mean ± SEM. p-values were calculated using one-way analysis of variance (ANOVA) followed by Dunnett’s multiple-comparison test against no UAS control. NS, p ≧ 0.05; †, p < 0.05. Sample sizes are shown in the figure. (H) Representative images of female wing upon Bruce or Bruce-RNAi (Bruce-i) overexpression using En-Gal4. The grey area (labelled as A) represents the anterior part, and the green area (labelled as P) represents the posterior part of the wing. Scale bar: 1 mm. (I) Quantification of relative posterior part of wing size normalized to corresponding anterior part of wing size. Data are presented as mean ± SEM. p-values were calculated using one-way ANOVA followed by Dunnett’s multiple comparison test against no UAS control. †, p < 0.05. Sample sizes are shown in the figure. (J) Representative images of female wing upon InRDN and Bruce-RNAi (Bruce-i) overexpression using WP-Gal4. Scale bar: 1 mm. (K) Quantification of relative wing size normalized to control (WP > +). Data are presented as mean ± SEM. p-values were calculated using one-way ANOVA followed by Sidak’s multiple comparison test for selected pairs.†, p < 0.05. Sample sizes are shown in the figure. (L) Schematic diagram of Dcp-1 activity-regulating alternative apoptosis signaling pathway in Drosophila mediated by autophagy-Bruce axis.

Dcp-1 overexpression induces apoptosis in Drosophila S2 cells.

(A) Representative images of Drosophila S2 cells transfected with mNeonGreen (mNG)-tagged wild-type (WT) or catalytically inactive (catalytic cysteine mutated to glycine; CG) Dcp-1 and Drice. Scale bars: 50 µm (left) and 5 µm (right). (B) Quantification of mNG intensity for each cell, shown as a violin plot. Quartiles are indicated by black broken lines, and the median is indicated by a red bar. (B′) Magnified view of the data in (B), with the Y-axis limited to 0–40. p-values were calculated using one-way analysis of variance (ANOVA) followed by Sidak’s multiple-comparison test for selected pairs. NS, p > 0.05; †, p < 0.05. Sample sizes: mNG (n = 712 cells), Dcp-1WT::mNG (n = 212 cells), Dcp-1CG::mNG (n = 380 cells), DriceWT::mNG (n = 434 cells), and DriceCG::mNG (n = 442 cells). (C) Quantification of the average mNG intensity in each field of view. Data are presented as mean ± SEM. p-values were calculated using one-way ANOVA followed by Sidak’s multiple-comparison test for selected pairs. NS, p ≧ 0.05; †, p < 0.05. Sample size for each condition is 4. (D) Quantification of the mNG-positive cell number in each field of view. Data are presented as mean ± SEM. p-values were calculated using one-way ANOVA followed by Sidak’s multiple-comparison test for selected pairs. NS, p ≧ 0.05; †, p < 0.05. Sample size for each condition is 4.

Expression patterns of Drosophila caspases in larval and adult tissues.

(A) Western blotting of expression of C-terminally V5::TurboID knocked-in tagged caspases and biotinylation patterns of their proximal proteins detected by streptavidin (SA) in larval salivary gland, fat body, and brain. (B) Western blotting of expression of C-terminally V5::TurboID knocked-in tagged caspases and biotinylation patterns of their proximal proteins detected by SA in adult thorax, ovary, and testis.

RNAi screening of Dcp-1-enriched proximal proteins for regulators of Dcp-1 activation.

(A) Quantification of female wing phenotypes upon knockdown of Dcp-1-enriched proximal proteins driven by WP-Gal4. “XX” denotes the knocked-down gene, and “#1” and “#2” indicate independent RNAi lines. Each wing is manually classified into four (wingless, severe, mild, and intact) categories. Sample sizes are shown in the figure. (B) Quantification of female wing phenotypes upon Dcp-1::VENUS overexpression driven by WP-Gal4 with simultaneous knockdown of Dcp-1-enriched proximal proteins. “XX” denotes the knocked-down gene, and “#1” and “#2” indicate independent RNAi lines. Each wing is manually classified into four (wingless, severe, mild, and intact) categories. Sample sizes are shown in the figure.

Predicted structures of Bruce complexed with Dcp-1 and Drice IBM-like sequences.

(A) Overall view of the predicted complex structure of Bruce and Dcp-1. Model 0 is shown with the same color scheme as in Figure 4A and B. The predicted position error is shown in (A’). (B) Overall view of the predicted complex structure of Bruce and Drice. Model 0 is shown with the same color scheme as in Fig. 4A and C. The predicted position error is shown in (B’). (C) Structural comparison between the experimentally determined complex of XIAP BIR3 and Smac IBM (C), and the predicted complex of dBruce BIR and Dcp-1 IBM (C’). XIAP BIR3 is shown as a pink cartoon and the Smac IBM as green sticks (PDB ID: 1G73, right panel). dBruce BIR domain is shown as a pink cartoon and the Dcp-1 IBM as dark green sticks (model 0, left panel). Zn²⁺ ions are shown as gray spheres. Salt bridges are indicated by black dashed lines, along with the corresponding interacting residues.

Expression patterns of mStayGold::V5-tag knocked-in Bruce.

(A) Schematic diagram of CRISPR/Cas9-mediated knock-in of mStayGold::C4 linker (C4)::V5 tag at the N-terminus of Bruce. A 3xP3-DsRed cassette was integrated at the endogenous TTAA site as a transformation marker. Removal of the cassette restores the genomic TTAA site, enabling scarless genome engineering. (B) Expression pattern of mStayGold (mSG)::C4::V5-tag knocked-in Bruce (green) in the ovariole. Nuclei are visualized using Hoechst 33342 (magenta). Scale bar: 50 µm. (C) Expression pattern of mSG::C4::V5-tag knocked-in Bruce (green) in the testis. Nuclei are visualized using Hoechst 33342 (magenta). Scale bar: 50 µm. (D) Western blotting of expression of mSG::C4::V5-tag knocked-in tagged Bruce in the whole adult male and female. (E) Expression pattern of mSG::C4::V5-tag knocked-in tagged Bruce (green) in the wing imaginal discs. Nuclei are visualized using Hoechst 33342 (magenta). Scale bar: 50 µm. (F) Expression pattern of mSG::C4::V5-tag knocked-in tagged Bruce (green) in the wing imaginal discs upon Bruce-RNAi using WP-Gal4. Nuclei are visualized using Hoechst 33342 (magenta). Scale bar: 50 µm.