The molecular mechanism and evolutionary divergence of caspase 3/7-regulated gasdermin E activation
- CAS and Shandong Province Key Laboratory of Experimental Marine Biology, Institute of Oceanology; CAS Center for Ocean Mega-Science, Chinese Academy of Sciences, China
- Laboratory for Marine Biology and Biotechnology, Qingdao Marine Science and Technology Center, China
- College of Marine Sciences, University of Chinese Academy of Sciences, China
Figures
Figure 1 with 3 supplements
Cleavage of TrGSDME by caspases.
(A, B) TrGSDME was treated with 1 U of different HsCASPs for 2 hr and then subjected to SDS-PAGE (A) and immunoblotting (B). (C) SDS-PAGE analysis of purified TrCASP3/7. The p10 and p20 subunits are …
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Figure 1—source data 1
Original file for the western blot or SDS-PAGE analysis in Figure 1.
- https://cdn.elifesciences.org/articles/89974/elife-89974-fig1-data1-v1.zip
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Figure 1—source data 2
File containing Figure 1A–C and F–J and original scans of the relevant western blot or SDS-PAGE analysis with highlighted bands.
- https://cdn.elifesciences.org/articles/89974/elife-89974-fig1-data2-v1.zip
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Figure 1—source data 3
Numerical data from all bar graphs shown in Figure 1.
- https://cdn.elifesciences.org/articles/89974/elife-89974-fig1-data3-v1.zip
Figure 1—figure supplement 1
Phylogenetic analysis of the TrGSDME used in this study.
The phylogenetic tree was constructed using 15 fish GSDMEa (red) and 51 fish GSDMEb (blue) collected from the NCBI databank. The TrGSDME used in this study is indicated by a red star.
Figure 1—figure supplement 2
Cleavage of TrGSDME mutants by HsCASP3/7.
TrGSDME wild type (WT) and mutants (D255R and D258A) were treated with or without (-) HsCASP3/7 for 2 hr, and the cleavage was determined by immunoblotting with anti-His antibody. FL, full length; …
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Figure 1—figure supplement 2—source data 1
Original file for the western blot analysis in Figure 1—figure supplement 2.
- https://cdn.elifesciences.org/articles/89974/elife-89974-fig1-figsupp2-data1-v1.zip
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Figure 1—figure supplement 2—source data 2
File containing Figure 1—figure supplement 2 and original scans of the relevant western blot analysis with highlighted bands.
- https://cdn.elifesciences.org/articles/89974/elife-89974-fig1-figsupp2-data2-v1.zip
Figure 1—video 1
TrGSDME-NT induces pyroptosis of HEK293T cells.
HEK293T cells were transfected with the vector expressing mCherry-tagged TrGSDME-NT, and time-lapse images of the cells were recorded with a confocal microscope.
Figure 2 with 3 supplements
The pyroptotic activity of TrGSDME.
(A–C) HEK293T cells were transfected with C-terminally mCherry-tagged TrGSDME FL or truncate (NT or CT) for 24 hr, and then analyzed for TrGSDME expression (A), morphological change (B), and lactate …
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Figure 2—source data 1
Original file for the western blot analysis in Figure 2.
- https://cdn.elifesciences.org/articles/89974/elife-89974-fig2-data1-v1.zip
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Figure 2—source data 2
File containing Figure 2I and original scans of the relevant western blot analysis with highlighted bands.
- https://cdn.elifesciences.org/articles/89974/elife-89974-fig2-data2-v1.zip
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Figure 2—source data 3
Numerical data from all bar graphs shown in Figure 2.
- https://cdn.elifesciences.org/articles/89974/elife-89974-fig2-data3-v1.zip
Figure 2—figure supplement 1
Ectopic expression of TrGSDME-FL and truncates.
HEK293T cells were transfected with the backbone vector or the vector expressing mCherry-tagged TrGSDME full length (FL) or truncates (NT or CT) for 24 h. The cell lysate was then immunoblotted with …
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Figure 2—figure supplement 1—source data 1
Original file for the western blot analysis in Figure 2—figure supplement 1.
- https://cdn.elifesciences.org/articles/89974/elife-89974-fig2-figsupp1-data1-v1.zip
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Figure 2—figure supplement 1—source data 2
File containing Figure 2—figure supplement 1 and original scans of the relevant western blot analysis with highlighted bands.
- https://cdn.elifesciences.org/articles/89974/elife-89974-fig2-figsupp1-data2-v1.zip
Figure 2—figure supplement 2
The cell death-inducing ability of TrGSDME-FL and truncates.
HEK293T cells were transfected with the backbone vector or the vector expressing mCherry-tagged TrGSDME full length (FL) or truncate (NT or CT) for 24 hr. The cells were then stained with Sytox …
Figure 2—figure supplement 3
Activation of TrGSDME-mediated pyroptosis by TrCASP3/7.
(A) HEK293T cells were transfected with the backbone vector or the vector expressing the indicated protein for 24 hr and then stained with Sytox Green. Scale bars, 30 μm. (B, C) HEK293T cells …
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Figure 2—figure supplement 3—source data 1
Original file for the western blot analysis in Figure 2—figure supplement 3.
- https://cdn.elifesciences.org/articles/89974/elife-89974-fig2-figsupp3-data1-v1.zip
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Figure 2—figure supplement 3—source data 2
File containing Figure 2—figure supplement 3C and original scans of the relevant western blot analysis with highlighted bands.
- https://cdn.elifesciences.org/articles/89974/elife-89974-fig2-figsupp3-data2-v1.zip
Figure 3 with 2 supplements
GSDME-CT domain determines the recognition by CASP7.
(A) HsGSDME was treated with 1 U of various HsCASPs for 2 hr, and the products were analyzed by immunoblotting with anti-HsGSDME-CT antibody. (B) HsCASP3/7 and TrCASP3/7 were incubated with …
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Figure 3—source data 1
Original file for the western blot analysis in Figure 3.
- https://cdn.elifesciences.org/articles/89974/elife-89974-fig3-data1-v1.zip
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Figure 3—source data 2
File containing Figure 3A, D–F, and I and original scans of the relevant western blot analysis with highlighted bands.
- https://cdn.elifesciences.org/articles/89974/elife-89974-fig3-data2-v1.zip
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Figure 3—source data 3
Numerical data from the bar graph shown in Figure 3.
- https://cdn.elifesciences.org/articles/89974/elife-89974-fig3-data3-v1.zip
Figure 3—figure supplement 1
Cleavage of HsGSDME by caspases.
Recombinant HsGSDME was incubated with or without (-) HsCASP1, 2, 3, 6, 7, 8, and 9 for 2 hr, and the cleavage was determined by SDS-PAGE. M, molecular markers. FL, full length; NT, N-terminal …
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Figure 3—figure supplement 1—source data 1
Original file for the SDS-PAGE analysis in Figure 3—figure supplement 1.
- https://cdn.elifesciences.org/articles/89974/elife-89974-fig3-figsupp1-data1-v1.zip
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Figure 3—figure supplement 1—source data 2
File containing Figure 3—figure supplement 1 and original scans of the relevant SDS-PAGE analysis with highlighted bands.
- https://cdn.elifesciences.org/articles/89974/elife-89974-fig3-figsupp1-data2-v1.zip
Figure 3—figure supplement 2
Proteolytic activity of TrCASP1.
TrCASP1 was incubated with colorimetric Ac-YVAD-ρNA, Ac-DAVD-ρNA, and Ac-DMPD-ρNA for 1 hr, and the cleavage was determined by measuring the release of ρNA. n=3. Values are the means of triplicate …
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Figure 3—figure supplement 2—source data 1
Numerical data from the bar graph shown in Figure 3—figure supplement 2.
- https://cdn.elifesciences.org/articles/89974/elife-89974-fig3-figsupp2-data1-v1.zip
Figure 4 with 1 supplement
The p10 subunit of CASP7 is essential for discrimination on GSDME.
(A) Sequence alignment of HsCASP7 and TrCASP7 with HsCASP7 (PDB:1K86) as the template. α-helices and β-strands are indicated. The motifs involved in catalytic reaction (SHG and QACRG) and substrate …
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Figure 4—source data 1
Original file for the western blot analysis in Figure 4.
- https://cdn.elifesciences.org/articles/89974/elife-89974-fig4-data1-v1.zip
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Figure 4—source data 2
File containing Figure 4D–G and original scans of the relevant western blot analysis with highlighted bands.
- https://cdn.elifesciences.org/articles/89974/elife-89974-fig4-data2-v1.zip
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Figure 4—source data 3
Numerical data from the bar graph shown in Figure 4.
- https://cdn.elifesciences.org/articles/89974/elife-89974-fig4-data3-v1.zip
Figure 4—figure supplement 1
Preparation of recombinant proteins.
(A) SDS-PAGE analysis of purified Hsp20-Trp10 and Trp20-Hsp10. (B, C) Purified TrCASP7 (B) and HsCASP7 (C) were subjected to SDS-PAGE (upper panel) and immunoblotting with anti-His tag antibody …
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Figure 4—figure supplement 1—source data 1
Original file for the western blot or SDS-PAGE analysis in Figure 4—figure supplement 1.
- https://cdn.elifesciences.org/articles/89974/elife-89974-fig4-figsupp1-data1-v1.zip
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Figure 4—figure supplement 1—source data 2
File containing Figure 4—figure supplement 1A–E and original scans of the relevant western blot or SDS-PAGE analysis with highlighted bands.
- https://cdn.elifesciences.org/articles/89974/elife-89974-fig4-figsupp1-data2-v1.zip
Figure 5 with 3 supplements
Functional importance of the non-conserved residues in the p10 of HsCASP7 and TrCASP7.
(A) Sequence alignment of the p10 region in HsCASP7 and TrCASP7. The arrows indicate non-conserved residues. (B, C) HsGSDME was treated with wild type (WT) or mutant HsCASP7 (B) or TrCASP7 (C) for 2 …
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Figure 5—source data 1
Original file for the western blot analysis in Figure 5.
- https://cdn.elifesciences.org/articles/89974/elife-89974-fig5-data1-v1.zip
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Figure 5—source data 2
File containing Figure 5B–K and original scans of the relevant western blot analysis with highlighted bands.
- https://cdn.elifesciences.org/articles/89974/elife-89974-fig5-data2-v1.zip
Figure 5—figure supplement 1
Cleavage of poly (ADP-ribose) polymerase 1 (PARP1) and gelsolin by HsCASP7.
HEK293T cells were transfected with C-terminal Flag-tagged PARP1 (left panel) or gelsolin (right panel) for 24 hr. The cell lysates were incubated with or without (-) different units of HsCASP7 and …
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Figure 5—figure supplement 1—source data 1
Original file for the western blot analysis in Figure 5—figure supplement 1.
- https://cdn.elifesciences.org/articles/89974/elife-89974-fig5-figsupp1-data1-v1.zip
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Figure 5—figure supplement 1—source data 2
File containing Figure 5—figure supplement 1 and original scans of the relevant western blot analysis with highlighted bands.
- https://cdn.elifesciences.org/articles/89974/elife-89974-fig5-figsupp1-data2-v1.zip
Figure 5—figure supplement 2
Structural analysis of HsCASP1/4/7.
(A) The structures of HsCASP1 (PDB: 6kn0, gray), HsCASP4 (PDB: 6kmz, green), and HsCASP7 (PDB: 6kn0, purple) are superimposed. The βIII/βIII’ sheets of HsCASP1/4 are indicated by arrows; the …
Figure 5—figure supplement 3
GSDME cleavage by CASP7 in cellular system.
TrGSDME (A) or HsGSDME (B) was co-expressed with different doses of TrCASP7/HsCASP7 for 48 hr in HEK293T cells. The expression/cleavage of TrGSDME/HsGSDME and the expression of TrCASP7/HsCASP7 were …
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Figure 5—figure supplement 3—source data 1
Original file for the western blot analysis in Figure 5—figure supplement 3.
- https://cdn.elifesciences.org/articles/89974/elife-89974-fig5-figsupp3-data1-v1.zip
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Figure 5—figure supplement 3—source data 2
File containing Figure 5—figure supplement 3 and original scans of the relevant western blot analysis with highlighted bands.
- https://cdn.elifesciences.org/articles/89974/elife-89974-fig5-figsupp3-data2-v1.zip
Figure 6 with 2 supplements
Conservation and functional importance of S/N234 in CASP3/7.
(A) Weblogo analysis of the conservation of S/N234 in CASP3/7 in Mammalia, Aves, Reptilia, Amphibia, and Osteichthyes. (B) Sequence alignment of HsCASP3/7. The S/N residues are indicated by red …
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Figure 6—source data 1
Original file for the western blot analysis in Figure 6.
- https://cdn.elifesciences.org/articles/89974/elife-89974-fig6-data1-v1.zip
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Figure 6—source data 2
File containing Figure 6C, D–G, and I and original scans of the relevant western blot analysis with highlighted bands.
- https://cdn.elifesciences.org/articles/89974/elife-89974-fig6-data2-v1.zip
Figure 6—figure supplement 1
Conservation of S/N234 in mammalian CASP7.
The four conserved amino acids are shown in LOGOs, with height representing the relative proportion of the corresponding amino acid at that position. The S/N234 is presented as the fourth LOGO and …
Figure 6—figure supplement 2
The cleavage of MmGSDME by MmCASP3.
(A) SDS-PAGE analysis of purified MmCASP3. (B) MmGSDME was treated with 1 U of MmCASP3 for 2 hr and then subjected to SDS-PAGE.
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Figure 6—figure supplement 2—source data 1
Original file for the SDS-PAGE analysis in Figure 6—figure supplement 2.
- https://cdn.elifesciences.org/articles/89974/elife-89974-fig6-figsupp2-data1-v1.zip
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Figure 6—figure supplement 2—source data 2
File containing Figure 6—figure supplement 2 and original scans of the relevant SDS-PAGE analysis with highlighted bands.
- https://cdn.elifesciences.org/articles/89974/elife-89974-fig6-figsupp2-data2-v1.zip
Author response image 1
HsCASP7 activation by raptinal.
HEK293T cells were transfected with the empty vector (-), or the vector expressing HsCASP7 or HsCASP7-S234N for 24 h. The cells were then treated with or without (control) 5 μM raptinal for 4 h. The …
Author response image 2
The three-dimensional structure model of HsGSDME.
(A) The structure of HsGSDME was modeled by using mouse GSDMA3 (MmGSDMA3) as the template. The N-terminal domain (1-246 aa) and the C-terminal domain (279-468 aa) of HsGSDME are shown in red and …
Author response image 3
Conserved domain analysis of mouse (upper) and human (lower) GSDME.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Takifugu rubripes) | TrGSDME | GenBank | XP_029701077.1 | |
| Strain, strain background (Escherichia coli) | Trelief 5a | Tsingke Biological Technology | Cat#TSC01 | |
| Strain, strain background (E. coli) | Transetta (DE3) | TransGen | Cat#CD801 | Protein purification |
| Cell line (Homo sapiens) | HEK293T | ATCC | Cat#CRL-3216 | |
| Transfected construct (human) | Lipofectamine 3000 | Invitrogen | Cat#L3000075 | |
| Antibody | Anti-GSDME (rabbit monoclonal) | Abcam | Cat#ab215191 | WB (1:2000) |
| Antibody | Anti-GSDME (rabbit monoclonal) | Abcam | Cat#ab221843 | WB (1:2000) |
| Antibody | Anti-Flag (mouse polyclonal) | Abclonal | Cat#AE005 | WB (1:5000) |
| Antibody | Anti-Myc (mouse polyclonal) | Abclonal | Cat#AE010 | WB (1:5000) |
| Antibody | Anti-His (mouse polyclonal) | Abclonal | Cat#AE003 | WB (1:5000) |
| Antibody | Anti-mCherry (mouse polyclonal) | Abclonal | Cat#AE002 | WB (1:5000) |
| Antibody | Anti-TrGSDME (mouse polyclonal) | This paper | Against TrGSDME (1:1000 for WB) | |
| Recombinant DNA reagent | pmCherry-N1-TrGSDME variants (plasmid) | This paper | Transient expression (backbone: pmCherry-N1) | |
| Recombinant DNA reagent | pCMV-C-Myc-CASP3/7 (plasmid) | This paper | Transient expression (backbone: pCMV-C-Myc) | |
| Recombinant DNA reagent | pET-30a (+)-TrGSDME (plasmid) | This paper | Protein purification (backbone: pET-30a) | |
| Recombinant DNA reagent | pET-30a (+)-CASPs (plasmid) | This paper | Protein purification (backbone: pET-30a) | |
| Recombinant DNA reagent | pET-28a (+)-SUMO-HsGSDME (plasmid) | This paper | Protein purification (backbone: pET-28a) | |
| Recombinant DNA reagent | pET-28a (+)-SUMO-MmGSDME (plasmid) | This paper | Protein purification (backbone: pET-28a) | |
| Recombinant DNA reagent | pET-28a (+)-SUMO-GSDME chimeras (plasmid) | This paper | Protein purification (backbone: pET-28a) | |
| Sequence-based reagent | Primers | This paper | Supplementary file 1e | |
| Peptide, recombinant protein | HsCASP1 | Enzo Life Sciences | Cat#ALX-201-056 | |
| Peptide, recombinant protein | HsCASP2 | Enzo Life Sciences | Cat#ALX-201-057 | |
| Peptide, recombinant protein | HsCASP3 | Enzo Life Sciences | Cat#ALX-201-059 | |
| Peptide, recombinant protein | HsCASP6 | Enzo Life Sciences | Cat#ALX-201-060 | |
| Peptide, recombinant protein | HsCASP7 | Enzo Life Sciences | Cat#ALX-201-060 | |
| Peptide, recombinant protein | HsCASP8 | Enzo Life Sciences | Cat#ALX-201-060 | |
| Peptide, recombinant protein | HsCASP9 | Enzo Life Sciences | Cat#ALX-201-047 | |
| Chemical compound, drug | Ac-YVAD-ρNA | Enzo Life Sciences | Cat#ALX-260-026 | |
| Chemical compound, drug | Ac-VDQQD-ρNA | Sigma-Aldrich | Cat#SCP0105 | |
| Chemical compound, drug | Ac-DEVD-ρNA | Enzo Life Sciences | Cat#ALX-260-033 | |
| Chemical compound, drug | Ac-IETD-ρNA | Enzo Life Sciences | Cat#ALX-260-045 | |
| Chemical compound, drug | Ac-LEHD-ρNA | Enzo Life Sciences | Cat#ALX-260-081 | |
| Chemical compound, drug | Ac-DAVD-ρNA | This paper | Activity comparison of CASP3/7 | |
| Chemical compound, drug | Ac-DMPD-ρNA | This paper | Activity comparison of CASP3/7 | |
| Commercial assay or kit | CytoTox 96 Non-Radioactive Cytotoxicity Assay | Promega | Cat#G1780 | LDH release |
| Commercial assay or kit | Hieff Mut Site-Directed Mutagenesis Kit | Yeasen | Cat#11003ES10 | |
| Commercial assay or kit | FastPure Cell/Tissue Total RNA Isolation Kit V2 | Vazyme | Cat#RC112-01 | |
| Commercial assay or kit | Revert Aid First Strand cDNA Synthesis Kit | Thermo Fisher Scientific | Cat#K1622 | |
| Commercial assay or kit | Endotoxin-free plasmid kit | Sparkjade | Cat#AD0105 | |
| Software, algorithm | Prism software | GraphPad | https://www.graphpad.com/scientific-software/prism/ | |
| Other | Propidium iodide (PI) | Invitrogen | Cat#P1304MP | Cell death examination |
| Other | DiO | Solarbio | Cat#C1038 | Cell death examination |
Additional files
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Supplementary file 1
Supplementary tables.
(a) The CASP3 sequences used for WebLogo analysis in this study. (b) The CASP7 sequences used for WebLogo analysis in this study. (c) The credits for the pictures used in this study. (d) The constructs of the truncate and chimeric proteins. (e) The primers used in this study.
- https://cdn.elifesciences.org/articles/89974/elife-89974-supp1-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/89974/elife-89974-mdarchecklist1-v1.docx
