Caenorhabditis elegans caspase homolog CSP-2 inhibits CED-3 autoactivation and apoptosis in germ cells

Abstract

In Caenorhabditis elegans, apoptosis in germ cells is mediated by the same core apoptotic machinery that controls apoptosis in somatic cells. These include the CED-3 caspase, the CED-3 activator CED-4, and the cell death inhibitor CED-9. However, germline apoptosis also differs from somatic apoptosis in its regulation. We found that CSP-3, a caspase homolog that blocks CED-3 autoactivation and apoptosis in somatic cells, does not affect apoptosis in germ cells. Interestingly, the second C. elegans caspase homolog, CSP-2, shares sequence similarity to both catalytic subunits of the CED-3 caspase, and surprisingly, contains a stretch of sequence that is almost identical to that of CSP-3. Unlike CSP-3 that acts specifically in somatic cells, loss of CSP-2 causes increased apoptosis only in germ cells, suggesting that CSP-2 is a germ cell-specific apoptosis inhibitor. Moreover, like CSP-3, CSP-2 associates with the CED-3 zymogen and inhibits its autoactivation, but does not inhibit CED-4-induced CED-3 activation or the activity of the activated CED-3 protease. Thus, two different C. elegans caspase homologs use the same mechanism to prevent caspase autoactivation and apoptosis in different tissues, suggesting that this could be a generally applicable strategy for regulating caspase activation and apoptosis.

Figure 1

CSP-2, a CED-3 homologue, is expressed specifically in hermaphrodite germ cells. (a) Sequence alignment of CED-3, CSP-2B, and CSP-3. Residues that are identical are shaded in yellow and residues that are similar in pink. Trp131, Leu132, and Phe 186 of CSP-2B are indicated with red arrowheads. Underlines delineate the large and small subunits of CED-3, respectively. The box indicates the active-site pentapeptide of CED-3. (b) The csp-2 gene structure and deletion mutations. Exons are depicted as boxes and introns as lines. The translated regions of csp-2 are highlighted in blue. Three red boxes indicate the regions of csp-2 removed by the three csp-2 deletions, respectively. SL1 stands for SL1 trans-spliced leader sequence. (c) RT-PCR analysis of the relative abundance of CSP-2A and CSP-2B transcripts. Reverse transcription was performed on poly(A)_n mRNA isolated from mix-stage wild-type animals, followed by PCR amplification with primers specific to the CSP-2A coding region (PCR1 in b), or primers in the CSP-2A/2B coding region (PCR2 in b), or primers specific for a control gene rpl-26 (encoding a large ribosomal subunit L26 protein). In the PCR1 lane, the predicted 706-bp CSP-2A-specific RT-PCR product was not seen. In the PCR2 lane, amplification of the 622-bp product corresponding to the CSP-2A/2B transcript was detected. (d) Western blot analysis of P_csp-2csp-2::3xFlag transgenic animals. 25 P_csp-2csp-2::3xFlag transgenic hermaphrodites or males were solubilized in SDS sampling buffer and resolved on 15% SDS polyarylamide gel. The CstF-64 protein was used as a loading control. (e) csp-2 is specifically expressed in C. elegans germ cells. Differential interference contrast (DIC) and anti-GFP immuno-staining images of exposed gonads of wild type (N2) and smIs372 (P_csp-2csp-2::gfp) hermaphrodite animals are shown.

Figure 2

Inactivation of csp-2 does not affect apoptosis in somatic cells. (a, b) Embryonic cell corpse assays. Cell corpses were scored in the indicated strains. The ced-6(n2095) allele was used in b. Bean (B/C), 1.5-fold (1.5), 2-fold (2), 2.5-fold (2.5), 3-fold (3) and 4-fold (4) stage embryos were scored. The y axis represents the average number of cell corpses scored. Error bars are standard errors of mean (s.e.m.) At least 15 embryos were scored for each stage. The significance of differences between different genetic backgrounds was determined by unpaired t tests. *, P < 0.05; **, P < 0.0001. (c) Inactivation of csp-2 does not cause ectopic death of touch receptor neurons. An integrated transgene (bzIs8) was used to monitor the survival of six touch receptor neurons (green circles) as described in Materials and Methods. The percentages of animals missing one or more touch cells are shown. At least 100 animals were scored for each strain.

Figure 3

Loss of csp-2 causes increased apoptosis in germ cells. Germ cell corpses were scored in the indicated strains from one gonad arm 48 hours post L4 to adult molt (a, c, e, f and g) or every 12 hours post L4 to adult molt (b and d). Average numbers of germ cell corpses are shown. Error bars represent the standard errors of mean (s.e.m). At least 15 animals were scored in each strain or time point. In b, the significance of difference between ced-6(n2095) and csp-2(tm3077); ced-6(n2095) animals was determined by two-way analysis of variance (ANOVA). *, P < 0.05 for the whole time course. In c, the significance of differences between various csp; ced-6(n2095) strains and the ced-6(n2095) strain was determined by unpaired t tests. **, P < 0.0001. All other points had P values > 0.05. In d, the significance of difference between ced-2(n1994) and ced-2(n1994) csp-2(tm3077) animals was determined by two-way ANOVA. **, P < 0.0001 for the whole time course. In e, the significance of differences between different csp; ced-2(n1994) strains and the ced-2(n1994) strain was determined by unpaired t tests. **, P < 0.0001. All other points had P values > 0.05. (f, g) Rescue of the csp-2 mutant by P_pie-1gfp::csp-2B, P_csp-2csp-2::gfp, or P_hspcsp-2B transgenes. The significance of differences between transgenic strains and the ced-6(n2095); csp-2(tm3077) strain (f), between transgenic strains and the ced-2(n1994) csp-2(tm3077) strain (g), or between the smIs389-containing strains and the ced-2(n1994) strain (g) was determined by unpaired t tests. **, P < 0.0001. *, P < 0.01. All other points had P values > 0.05. (h) The P_hspcsp-2B transgenes rescue the missing cell defect of the csp-3(tm2260) mutant. The presence of touch cells was scored as described in Figure 2c.

Figure 4

CSP-2B associates with CED-3 in vitro. (a) CSP-2B binds to the CED-3 zymogen. GST-CSP-2B, GST-CSP-2B(mut), or GST was co-expressed in bacteria with the CED-3 zymogen tagged with a Flag epitode (CED-3-Flag). “mut” stands for W131E, L132R, and F186D substitutions. One portion of the soluble fraction was used in the western blot analysis to examine the expression levels of GST fusion proteins and CED-3-Flag using anti-GST and anti-Flag antibodies, respectively. The remaining portion of the soluble fraction was used in the GST fusion protein pulldown experiment and the amount of CED-3-Flag pulled down was analyzed by the western blot analysis using an anti-Flag antibody. (b) CSP-2B associates with both the large subunit and the small subunit of the CED-3 zymogen in vitro. The cartoon shows 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 large (p17) and the small (p13) subunits of CED-3 are shown below as boxes. The Flag epitode is labeled with green. GST-CSP-2B, GST-CSP-2B(F186D), GST-CSP-2B(W131E, L132R), GST-CSP-2B(C134E), or GST was co-expressed in bacteria with the CED-3 large subunit (Flag-p17) or the small subunit (p13-Flag), both of which are tagged with a Flag epitode. One portion of the soluble fraction was used in the western blot analysis to examine the expression levels of GST fusion proteins and CED-3 subunits using anti-GST and anti-Flag antibodies, respectively. The remaining portion of the soluble fraction was used in the GST fusion protein pulldown experiments and the amount of CED-3 subunits pulled down was analyzed by the western blot analysis using an anti-Flag antibody. (c) Structural model of the CED-3/CSP-2B complex. Because all caspases with known structures share a highly conserved core structure, the structure of caspase-3 (PDB code 1pau) was used to represent the structures of CED-3 and CSP-2B. The large subunit of CED-3 is shown in green, the small subunit shown in lemon. The large subunit of CSP-2B is shown in magenta, the small subunit in light pink.

Figure 5

CSP-2B specifically inhibits the autoactivation of the CED-3 zymogen in vitro. (a) CSP-2B inhibits autoactivation of the CED-3 zymogen. GST (93 nM, lanes 1–3), GST-CSP-2B (43 nM, lanes 4–6), or GST-CSP-2B(mut) (43 nM, lanes 7–9) were incubated with ³⁵S-Methionine labeled CED-3 zymogen as described in METHODS. At different time points (30, 90, and 150 min), an aliquot of the reaction was taken out and SDS sampling buffer was added to stop the reaction. The samples were resolved by 15% SDS polyacrylamide gel electrophoresis (SDS-PAGE) and subjected to autoradiography. (b) CSP-2B delays but does not block CED-4-mediated activation of CED-3. GST (93 nM) or GST-CSP-2B (43 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 30, 80, 130, 180, and 230 min, an aliquot of the reaction was taken out and SDS sampling buffer was added. The samples were resolved by 15% SDS-PAGE and subjected to autoradiography. (c) CSP-2B does not inhibit the activity of the active CED-3 protease in vitro. CED-3-Flag was co-expressed with GST or GST-CSP-2B for 3 hours in bacteria. The bacterial lysate containing similar levels of active CED-3 (acCED-3) and GST fusion proteins was incubated with ³⁵S-Methionine-labeled CED-9 for 2 hours at 30°C. In lane 4, the caspase inhibitor, iodoacetic acid (5 mM), was included. The reactions were resolved by 15% SDS-PAGE and detected by autoradiography. (d) A working model of how CSP-3 (left panel) and CSP-2B (right panel) inhibit CED-3 autoactivation and apoptosis in C. elegans somatic and germ cells, respectively.