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. 2008 Oct;15(10):1094-101.
doi: 10.1038/nsmb.1488. Epub 2008 Sep 7.

Inhibition of CED-3 zymogen activation and apoptosis in Caenorhabditis elegans by caspase homolog CSP-3

Xin Geng  1 , Yong Shi, Akihisa Nakagawa, Sawako Yoshina, Shohei Mitani, Yigong Shi, Ding Xue
Affiliations

Inhibition of CED-3 zymogen activation and apoptosis in Caenorhabditis elegans by caspase homolog CSP-3

Xin Geng et al. Nat Struct Mol Biol. 2008 Oct.

Abstract

Inhibitor of apoptosis (IAP) proteins have a crucial role in apoptosis, through negative regulation of caspases in species from fruitflies to mammals. In Caenorhabditis elegans, however, no IAP homolog or caspase inhibitor has been identified, calling into question how the cell-killing caspase CED-3 can be negatively regulated. Here we show that inactivation of the C. elegans csp-3 gene, which encodes a protein similar to the small subunit of the CED-3 caspase, causes cells that normally live to undergo apoptosis in a CED-3-dependent manner. Biochemical analysis reveals that CSP-3 associates with the large subunit of the CED-3 zymogen and inhibits zymogen autoactivation. However, CSP-3 does not block CED-3 activation induced by CED-4, nor does it inhibit the activity of the activated CED-3 protease. Therefore CSP-3 uses a previously unreported mechanism to protect cells from apoptosis.

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Figures

Figure 1
Figure 1. CSP-3 is a cytoplasmic protein with sequence homology to the small subunit of CED-3
(a) Sequence alignment of CSP-3 and the small subunits of CED-3 and human caspase-3. Residues that are identical are shaded in yellow and residues that are similar are labeled with pink. Phe57 of CSP-3 is indicated with a blue arrowhead. (b) The csp-3 gene structure and deletion mutations. Exons are depicted as boxes and introns as lines. The translated regions of csp-3 are highlighted in blue. Two orange boxes indicate the regions of csp-3 removed by the two csp-3 deletions, respectively. Also shown are a csp-3 translational gfp fusion and a 5.73-kb genomic rescuing fragment (csp-3(+)). (c) Both csp-3 deletions abolish expression of csp-3 in C. elegans. Immunoblotting for CSP-3 in total worm lysates from N2, csp-3(tm2260) and csp-3(tm2486) animals is shown. The CstF-64 protein was used as a loading control. (d) csp-3 is ubiquitously expressed in C. elegans embryos. Differential interference contrast (DIC) and GFP images of an early-stage embryo carrying Pcsp-3csp-3::gfp are shown.
Figure 2
Figure 2. Loss of csp-3 results in increased cell deaths during embryonic development
(a–c) Cell corpses were scored in the indicated strains. ced-5(n1812) and ced-6(n2096) alleles were used in b and c, respectively. Bean (B/C), 1.5-fold (1.5), 2.0-fold (2), 2.5-fold (2.5), 3.0-fold (3), 4.0-fold (4) –stage embryos and L1 larvae (L1) were examined. (d) Rescue of the csp-3 mutant by various transgenes. Four-fold stage embryos from csp-3(tm2260);ced-5(n1812) animals carrying the indicated transgenes were scored for cell corpses. In a–d, the y-axis represents the average number of cell corpses scored. Error bars are s.d. Fifteen embryos or larvae were scored for each stage or transgenic line. In a–c, the significance of differences between different genetic backgrounds was determined by analysis of variance and Fisher probable least-squares difference. *, P < 0.05; **, P < 0.0001. All other points had P values > 0.05. In d, data from two independent transgenic lines were analyzed similarly and compared with that of csp-3(tm2260);ced-5(n1812) animals. *, P < 0.05; **, P < 0.0001. (e) Western blot analysis of the expression levels of CSP-3 in two Psur-5csp-3 transgenic lines (line 1 and lines 2) compared with that of wild-type animals (N2) or csp-3(tm2260) animals. (f) Overexpression of CSP-3 in C. elegans mildly inhibits programmed cell death. Extra undead cells in the anterior pharynx were scored in two Psur-5csp-3 transgenic lines and two control Psur-5gfp transgenic lines. At least 15 transgenic animals were scored for each strain.
Figure 3
Figure 3. Loss of csp-3 causes cells that normally live to undergo programmed cell death
(a) Touch receptor neurons are randomly lost in csp-3 mutants via programmed cell death. An integrated transgene (bzIs8) was used to monitor the survival of six touch receptor neurons (green circles) as described in Methods. The percentage of a specific touch-cell type lost was shown. At least 100 animals were scored for each strain. ALML, anterior lateral microtubule cell left; ALMR, anterior lateral microtubule cell right; AVM, anterior ventral microtubule cell; PLML, posterior lateral microtubule cell left; PLMR, posterior lateral microtubule cell right; PVM, posterior ventral microtubule cell. (b) A PLM neuron labeled by GFP in a csp-3(tm2260);ced-5(n1812);bzIs8 larva undergoes ectopic cell death and adopts a raised-button–like morphology characteristic of apoptotic cells (indicated by an arrowhead). (c) The csp-3(+) transgene rescues the missing cell defect of the csp-3(tm2260) mutant. Touch cells were scored as described in a. The percentage of animals missing at least one touch cell is shown. Ex, extrachromosomal transgenic array. (d) Several different types of neurons are also randomly lost in csp-3 mutants. An integrated transgene (inIs179) was used to label 16 neurons (green circles) in all strains examined. The presence of these neurons in L4 larvae was scored by epifluorescence microscopy. The percentage of a specific cell type missing was calculated as follows: the number of cells missing in a cell type divided by the number of total cells expected in a cell type. The specific cell types are: two ADE neurons, six ventral cord neurons (VC), two HSN neurons, six phasmid (PH) neurons (two PHA, two PHB and two PHC). The percentage of animals missing at least one neuron is also shown. At least 100 animals were scored for each strain.
Figure 4
Figure 4. CSP-3 associates with CED-3 in vitro and in C. elegans
(a) CSP-3 binds to the CED-3 zymogen. GST–CSP-3, GST–CSP-3(F57D) or GST was coexpressed in bacteria with the CED-3 zymogen tagged with a Flag epitode (CED-3–Flag). One portion of the soluble fraction was analyzed by western blot (IB) to examine the expression levels of GST fusion proteins and CED-3–Flag. The remaining portion of the soluble fraction was used for GST protein pull-down experiment, and the amount of CED-3–Flag pulled down was analyzed by western blot analysis. (b) CSP-3 associates specifically with the large subunit of CED-3 in vitro. GST–CSP-3, GST–CSP-3(F57D) or GST was coexpressed in bacteria with the CED-3 large subunit (p17) or the small subunit (p13), both tagged with a Flag epitode (gray box). Analysis of expression levels as well as the amounts of two CED-3 subunits coprecipitated with GST fusion proteins was conducted as described in a. The diagram above depicts the domain structure of the CED-3 zymogen, with arrows indicating the three proteolytic cleavage sites that lead to the activation of the CED-3 zymogen. The three CED-3 cleavage products are shown below as boxes. (c) CSP-3 associates with CED-3 in C. elegans. Lysates from C. elegans animals expressing CED-3::GFP or CEH-30::GFP were prepared as described in Methods. One portion of the worm lysate was used in the western blot analysis to examine the expression levels of CSP-3 and GFP fusion proteins. The remaining portion of the lysate was incubated with a mouse anti-GFP monoclonal antibody and precipitated using Protein G Sepharose beads. The amount of the CSP-3 protein pulled down with the GFP fusion proteins was analyzed by western blot using purified anti–CSP-3 antibody. A small amount of full-length CED-3::GFP fusion was detected in the lysate (data not shown). The predominant species detected was CED-3::GFP fusion without its prodomain but containing both large and small subunits.
Figure 5
Figure 5. CSP-3 specifically inhibits the autoactivation of the CED-3 zymogen
(a) CSP-3 inhibits autoactivation of the CED-3 zymogen. GST (93 nM, lanes 1–3), GST–CSP-3 (12 nM, lanes 4–6) or GST–CSP-3(F57D) (12 nM, lanes 7–9) was incubated with 35S-methionine labeled CED-3 zymogen as described in Methods. At different time points, an aliquot was taken out and SDS sampling buffer was added to stop the reaction. The samples were resolved by 15% SDS-PAGE and subjected to autoradiography. (b) CSP-3 delays but does not block CED-4–mediated activation of CED-3. GST (93 nM) or GST–CSP-3 (12 nM) was incubated with 35S-methionine labeled CED-3 zymogen in the absence or presence of oligomeric CED-4 (40 nM) (added 20 min later). At different time points, an aliquot was taken out and SDS sampling buffer was added. One half of the aliquot was resolved by 15% SDS-PAGE and subjected to autoradiography (above). One-fourth of the aliquot was used for the immunoblotting analysis using an anti-GST antibody (below). (c) Oligomeric CED-4 overrides CSP-3 inhibition to induce CED-3 activation. Preformed GST-CSP-3–CED-3-Flag complexes (~70 nM of CED-3–Flag) were incubated either with buffer or with increasing concentrations of oligomeric or monomeric CED-4 at 30 °C for 30 min (Methods), before being resolved on 15% SDS-PAGE and detected with an anti-Flag antibody. (d) CSP-3 does not inhibit the activity of the active CED-3 protease in vitro. CED-3–Flag was coexpressed with GST, GST–CSP-3 or GST–CSP-3(F57D) for 3 h in bacteria. The bacterial lysate containing similar levels of active CED-3 (acCED-3) and GST fusion proteins (Supplementary Fig. 2b) was incubated with 35S-methionine–labeled CED-9 for 2 h at 30 °C. In one reaction, the caspase inhibitor iodoacetic acid (5 mM) was included. The reactions were resolved by 15% SDS-PAGE and detected by autoradiography. (e) A CSP-3 working model in C. elegans. CSP-3 associates with the CED-3 zymogen in all cells. In cells that normally live, CSP-3 prevents the CED-3 zymogen from dimerization and inadvertent autoactivation. In cells that are programmed to die, CED-4 oligomer overrides CSP-3 inhibition to induce the activation of the CED-3 zymogen via the proximity-induced dimerization model.
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References

    1. Steller H. Mechanisms and genes of cellular suicide. Science. 1995;267:1445–1449. - PubMed
    1. Thornberry NA, Lazebnik Y. Caspases: enemies within. Science. 1998;281:1312–1316. - PubMed
    1. Boatright KM, Salvesen GS. Mechanisms of caspase activation. Curr. Opin. Cell Biol. 2003;15:725–731. - PubMed
    1. Shi Y. Mechanisms of caspase activation and inhibition during apoptosis. Mol. Cell. 2002;9:459–470. - PubMed
    1. Budihardjo I, Oliver H, Lutter M, Luo X, Wang X. Biochemical pathways of caspase activation during apoptosis. Annu. Rev. Cell Dev. Biol. 1999;15:269–290. - PubMed

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