Tamura R et al. (2018), Starfish Apaf-1 activates effector caspase-3/9 ...

ECB-ART-46095

Sci Rep 2018 Jan 25;81:1611. doi: 10.1038/s41598-018-19845-6.

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Starfish Apaf-1 activates effector caspase-3/9 upon apoptosis of aged eggs.

Tamura R , Takada M , Sakaue M , Yoshida A , Ohi S , Hirano K , Hayakawa T , Hirohashi N , Yura K , Chiba K .

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Caspase-3-related DEVDase activity is initiated upon apoptosis in unfertilized starfish eggs. In this study, we cloned a starfish procaspase-3 corresponding to mammalian effector caspase containing a CARD that is similar to the amino terminal CARD of mammalian capsase-9, and we named it procaspase-3/9. Recombinant procaspase-3/9 expressed at 15 °C was cleaved to form active caspase-3/9 which has DEVDase activity. Microinjection of the active caspase-3/9 into starfish oocytes/eggs induced apoptosis. An antibody against the recombinant protein recognized endogenous procaspase-3/9 in starfish oocytes, which was cleaved upon apoptosis in aged unfertilized eggs. These results indicate that caspase-3/9 is an effector caspase in starfish. To verify the mechanism of caspase-3/9 activation, we cloned starfish Apaf-1 containing a CARD, a NOD, and 11 WD40 repeat regions, and we named it sfApaf-1. Recombinant sfApaf-1 CARD interacts with recombinant caspase-3/9 CARD and with endogenous procaspase-3/9 in cell-free preparations made from starfish oocytes, causing the formation of active caspase-3/9. When the cell-free preparation without mitochondria was incubated with inactive recombinant procaspase-3/9 expressed at 37 °C, DEVDase activity increased and apoptosome-like complexes were formed in the high molecular weight fractions containing both sfApaf-1 and cleaved caspase-3/9. These results suggest that sfApaf-1 activation is not dependent on cytochrome c.

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Genes referenced: LOC100892760 LOC115918798 LOC115919910 LOC115925415 LOC577219 LOC577728 LOC594261 LOC594349 mapk1

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	Figure 1. The sequence and the domain organization of caspase from starfish A. pectinifera. (a) Amino acid sequence of starfish caspase. The conserved active peptide region is underlined (green). The amino acids in red indicate the cleavage sites. (b) The domain organization of starfish caspase. The 1356 nt open reading frame (ORF) of starfish caspase encodes a protein of 452 aa, which exhibits a typical caspase-9 domain architecture containing amino terminal CARD (residues 1–92), large subunit (residues 180–318), and small subunit (residues 357–452).
	Figure 2. Expression of, and proteolytic activity assay for, recombinant caspase-3/9. (a) SDS-PAGE analysis of recombinant procaspase-3/9-His6 expressed in E. coli with CBB gel staining. Lanes: (1) No IPTG induction; (2) Procaspase-3/9 with IPTG induction at 37 °C; (3) Cleaved caspase-3/9 with IPTG induction at 15 °C. Full gel is presented in Supplementary Fig. S10. (b) Specific proteolytic activity of recombinant caspase-3/9-His6. Cell lysate from E. coli either transformed with a vector encoding caspase-3/9-His6 (casp) or control vector (vec) were analyzed for caspase-3 (DEVD), -8 (IETD), and -9 (LEHD) catalytic activity using Ac-DEVD-MCA, Ac-IETD-MCA and Ac-LEHD-MCA, respectively. (c) DEVDase activity of recombinant caspase-3/9-His6 expressed at different temperatures. Cell lysate from E. coli without IPTG induction, with IPTG induction at 37 °C, and with IPTG induction at 15 °C were analyzed for DEVDase activity using Ac-DEVD-MCA. (d) Microinjection of caspase-3/9-His6 into oocytes. Purified caspase-3/9-His6 (1.1 µg/mL at a final concentration) or control buffer was microinjected into immature oocytes, and photographs were taken at the indicated times after microinjection. (e) The number of apoptotic eggs was counted after microinjection of purified caspase-3/9-His6 (closed triangle, 1.1 µg/mL; open triangle, 0.56 µg/mL at a final concentration) or control buffer (circle). The results are representative of four independent experiments.
	Figure 3. Activation and cleavage of endogenous caspase-3/9 upon apoptosis in unfertilized eggs. (a) CBB gel staining and western blotting analysis of recombinant caspase-3/9-His6. Cell lysate of E. coli expressing recombinant caspase-3/9-His6 was subjected to SDS-PAGE, followed by CBB gel staining (left panel), or analyzed by western blotting using the anti-caspase-3/9 antibody (right panel). Lanes: (1) with induction of IPTG at 37 °C; (2) at 15 °C. (b) Time course of endogenous caspase-3/9 activation after 1-MA treatment. Samples of oocytes were analyzed by SDS-PAGE and western blotting with the anti- caspase-3/9 antibody. Cleaved caspase-3/9 was visible after longer exposures. At the same time, the activity of endogenous caspase-3/9 was measured by the cleavage of Ac-DEVD-MCA. The morphological changes of the oocytes/eggs were observed with a light microscope equipped with Nomarski differential interference contrast optics; (0:00) immature oocyte; (0:20–4:00) mature eggs; (8:20) blebbing egg; (9:30–11:00) fragmented eggs. (c) Dynamics of caspase-3/9, ERK1/2 and p38MAPK during apoptosis. Samples were analyzed by western blotting with anti-caspase-3/9, anti-ERK1/2, and active p38MAPK-specific antibodies. Full gel and blots are presented in Supplementary Fig. S10. The results are representative of three independent experiments.
	Figure 4. The amino acid sequence and the domain organization of sfApaf-1. (a) Amino acid sequence of sfApaf-1. The one putative nucleotide-binding site (GXXGXGK) is underlined (orange). sfApaf-1 contains three domains: CARD (red), NOD (purple), and 11 WD40 repeats (green). (b) Domain organization of sfApaf-1. The 3714 nt open reading frame (ORF) of starfish Apaf-1 encodes a 1238-aa protein with an amino terminal CARD (residues 13–90), NOD (residues 136–396), and WD40 repeat region (residues 621–1156).
	Figure 5. Interaction of sfApaf-1 CARD with caspase-3/9 CARD. (a) Glutathione Sepharose 4B bead-bound GST-A-CARD was incubated with His-C-CARD in PBS buffer; glutathione Sepharose 4B-bound GST was used as a control. Associated His-C-CARD was co-eluted with GST-A-CARD (right panel), but not with GST (left panel) after the addition of elution buffer, as determined by SDS–PAGE and CBB gel staining. (b) Glutathione magnetic agarose bead-bound GST-A-CARD or similarly bead-bound GST (control) was incubated with cell-free preparations containing endogenous procaspase-3/9. Endogenous procaspase-3/9 was pulled down with GST-A-CARD (right panel), but not with GST (left panel), and identified by western blotting with anti-caspase-3/9 antibody. Full blots are presented in Supplementary Fig. S10. The results are representative of two independent experiments.
	Figure 6. Activation of caspase-3/9 by sfApaf-1 CARD. (a) Activation of endogenous caspase-3/9 in cell-free preparation by GST-A-CARD. A time course of DEVDase activity was measured at the indicated time after addition of GST-A-CARD or control GST. (b) Cleavage of endogenous caspase-3/9 in cell-free preparations was induced by GST-A-CARD but not by GST, and detected by western blotting with the anti-caspase-3/9 antibody. (c) The interaction between A-CARD and caspase-3/9 in cell-free preparations. The cell-free preparation was incubated with GST-A-CARD or not, and fractionated by using gel filtration chromatography. Each fraction was analyzed by western blotting with anti-caspase-3/9 and anti-GST antibodies. (d) DEVDase activity in each fraction was measured by the cleavage of Ac-DEVD-MCA. The yellow column is from gel filtered cell-free preparations incubated with GST-A-CARD, and the blue column is from gel-filtered cell-free preparation without recombinant protein. Full blots are presented in Supplementary Fig. S10. The results are representative of three independent experiments.
	Figure 7. Apoptosome-like complex formation by recombinant procaspase-3/9-His6 in the cell-free preparations. (a) Western blot analysis of recombinant GST-sfApaf-1 CARD (1–134 aa) protein using anti-sfApaf-1 antibody. Lanes: (1) without IPTG induction; (2) with IPTG induction at 37 °C. (b) Ultracentrifuged cell-free preparations were fractionated by gel filtration chromatography. Endogenous procaspase-3/9 and sfApaf-1 were detected by western blotting with anti-caspase-3/9 and anti-sfApaf-1 antibodies. (c) Activation of endogenous caspase-3/9 in cell-free preparations by treatment with procaspase-3/9-His6. A time course of DEVDase activity was measured at the indicated times after adding either procaspase-3/9-His6 or buffer (control). (d) Ultracentrifuged cell-free preparations were incubated with recombinant procaspase-3/9-His6, and fractionated by gel filtration chromatography. Fractions were analyzed by western blotting with the anti-caspase-3/9 antibody and anti-sfApaf-1 antibody. (e) DEVDase activity in fractions was measured by the cleavage of Ac-DEVD-MCA. The red column is from gel filtered cell-free preparations with procaspase-3/9-His6, and the blue column is from gel filtered cell-free preparations without recombinant protein. Full blots are presented in Supplementary Fig. S10. The results are representative of two independent experiments.
	Figure 8. Structure of human Apaf-1 WD40 repeat in complex with horse cytochrome c and the characteristics of the interface. (a) Amino acid residues in WD40 repeats of vertebrate Apaf-1 that interact with horse cytochrome c and the corresponding residues in sfApaf-1. The residue in gray background has the same type of amino acid as in human Apaf-1. Note that sfApaf-1 has a long deletion in each propeller domain compared with Apaf-1 of other animals. (b) The three-dimensional structure of human Apaf-1 (WD40 repeat region) in complex with horse cytochrome c (PDB ID: 3jbt chains A and B). WD40 repeat region is colored from green to red and cytochrome c in black. Amino acid residues in WD40 repeat that interact with cytochrome c are depicted in stick model. The interaction Structure of human Apaf-1 WD40 repeat in complex with horse cytochrome c and the characteristics of the interface. The interaction was calculated based on ∆accessiblity and chose the residues that have difference in solvent accessible area, when the protein interacts with the partner or not. Two loops in gray protruding from WD40 repeat to cytochrome c are deleted in sfApaf-1. (c) Percentage identity in WD40 repeats (blue) and that in cytochrome c binding residues (orange) between Apaf-1 of human and other animals. Note that the values of percentage identity reverse in starfish Apaf-1. (d) The amino acid sequence alignment of cytochrome c from animals. The sequences were obtained from UniPort and the ID is shown at the end of each sequence. The sequence identities are between 73 (starfish and human) and 100 (rat and mouse) %, which are much higher than those in WD40 repeat of Apaf-1.

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