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Regulation of CED-3 caspase localization and activation by C. elegans nuclear-membrane protein NPP-14

Nature Structural & Molecular Biology volume 23, pages 958–964 (2016)Cite this article

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Abstract

Caspases are cysteine proteases with critical roles in apoptosis. The Caenorhabditis elegans caspase CED-3 is activated by autocatalytic cleavage, a process enhanced by CED-4. Here we report that the CED-3 zymogen localizes to the perinuclear region in C. elegans germ cells and that CED-3 autocatalytic cleavage is held in check by C. elegans nuclei and activated by CED-4. The nuclear-pore protein NPP-14 interacts with the CED-3 zymogen prodomain, colocalizes with CED-3 in vivo and inhibits CED-3 autoactivation in vitro. Several missense mutations in the CED-3 prodomain result in stronger association with NPP-14 and decreased CED-3 activation by CED-4 in the presence of nuclei or NPP-14, thus leading to cell-death defects. Those same mutations enhance autocatalytic cleavage of CED-3 in vitro and increase apoptosis in vivo in the absence of npp-14. Our results reveal a critical role of nuclei and nuclear-membrane proteins in regulating the activation and localization of CED-3.

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Figure 1: Three unique CED-3 prodomain mutations affect CED-3 autoactivation in vitro.
Figure 2: CED-3 localizes to the perinuclear region in C. elegans germ cells.
Figure 3: C. elegans nuclei inhibit CED-3 zymogen autoactivation in vitro.
Figure 4: NPP-14 interacts with the prodomain of CED-3 and consequently inhibits CED-3 zymogen activation.

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Acknowledgements

We thank C.L. Sun for generating the Pnpp-14npp-14::dsRed integrated transgene, X.C. Wang for isolating the sm160 allele, J. Friedman for identifying the sm160 mutation in npp-14, and Y.G. Shi (Tsinghua University) and M. Han (University of Colorado) for providing vectors. This work was supported by the National Basic Research Program of China (973 program 2013CB945600) and National Institutes of Health (NIH) grants T32GM007135 and F30NS070596 (to B.L.H.) and R01 GM59083, R01 GM88241 and R35 GM118188 (to D.X.).

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  1. Xudong Chen, Yue Wang and Yu-Zen Chen: These authors contributed equally to this work.

Authors and Affiliations

  1. School of Life Sciences, Tsinghua University, Beijing, China

    Xudong Chen, Yue Wang, Hongyan Guo & Ding Xue

  2. Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Tsinghua University, Beijing, China

    Xudong Chen & Ding Xue

  3. Department of Molecular, Cellular, and Developmental Biology, University of Colorado Boulder, Boulder, Colorado, USA

    Yu-Zen Chen, Brian L Harry, Akihisa Nakagawa, Eui-Seung Lee & Ding Xue

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Contributions

X.C., Y.W., Y.-Z.C. and D.X. conceived the research and designed and analyzed experiments. X.C. carried out all CED-3 in vitro assays and some immunoblotting and protein binding assays. Y.W. performed the RNAi screen, microscopy imaging, some protein binding assays and some of the somatic-cell death assays. Y.-Z.C. performed germ-cell death assays and some of the somatic-cell death assays, microscopy imaging and some biochemical assays. B.L.H., A.N., E.-S.L. and H.G. assisted in some experiments. X.C., Y.W., Y.-Z.C. and D.X. wrote the paper.

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Correspondence to Ding Xue.

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Integrated supplementary information

Supplementary Figure 1 Analysis of CED-3 autoactivation and CED-4-induced CED-3 activation.

(a) Repeat experiments of Fig. 1a. (b) Repeat experiments of Fig. 1b. (c) Quantitative analysis of three independent CED-3 autoactivation experiments shown in a and Fig. 1a and CED-4-induced CED-3 activation experiments shown in b and Fig. 1b. In each experiment, the combined intensity (Ia-t) of active CED-3 bands (17 kDa and 15 kDa bands very close together and indicated with red brackets) at the 25-min, 45-min, 60-min, or 90-min time point of each CED-3 protein (wild-type or mutant) and the intensity (Iz-25) of the CED-3 zymogen band at the 25-min time point were quantified using the Image J software (See Online Methods). The relative level of the CED-3 zymogen activation (Ca-t) at each time point (45-min, 60-min, or 90-min) in each experiments was determined as follow: Ca-t=(Ia-t-Ia-25)/Iz-25 (t indicates 45-min, 60-min, or 90-min time point). The average level of the CED-3 zymogen activation (Ca-t) at each time point was then determined from three independent experiments. Error bars are s.e.m. Compared with the corresponding Ca-t from wild-type CED-3, *** P < 0.001. ** P < 0.01. * P < 0.05 (two-sided t-tests). (d) Autoradiographs of SDS-PAGE showing time-course of activation of the CED-3 zymogen harboring a catalytic-site mutation, G360S, with or without oligomeric CED-4. Experiments were performed as described in Fig. 1.

Supplementary Figure 2 CED-3::GFP localization, purity of isolated C. elegans nuclei and CED-3 activity assays.

(a,b) The GFP localization pattern in C. elegans germ cells. Images of DIC, Hoechst, GFP and GFP/Hoechst merged from the exposed gonads of a wild-type hermaphrodite adult carrying a complex transgenic array containing the PhspGFP constructs (a) and a non-transgenic wild-type hermaphrodite adult (b) are shown. The animals were imaged 3 hours after the heat-shock treatment. Scale bars indicate 10 μm. (c) Representative DIC and GFP images from the exposed gonad of a hermaphrodite adult animal carrying the Pced-3ced-3::gfp integrated transgene are shown. The white arrowheads indicate live germ cells and the red arrowhead indicates an apoptotic germ cell. Scale bar indicates 10 μm. Insets show enlarged images of the apoptotic germ cell (upper) and a live germ cell indicated by the bottom white arrowhead. From nine dissected gonads of adult hermaphrodites, 13 out of 15 apoptotic germ cells show a similar diffuse CED-3::GFP pattern. (d) 5 μL of ccIs4810 nuclei or 10 μL of ccIs4810 worm lysate were resolved by 12% SDS-PAGE and detected by immunoblotting using antibodies to GFP (LMN-1::GFP, a nuclear membrane marker), α-tubulin (a cytoskeleton marker), DLG-1 (a plasma membrane marker), HSP-60 (a mitochondrial marker), and HDEL proteins (an ER marker), respectively. ccIs4810 is an integrated transgene that expresses a nuclear lamin GFP fusion. Purified nuclei were without cytoskeletal and other membrane contaminations. (e,f) Autoradiographs of SDS-PAGE showing CED-3 activity assays. 10 ng of the active CED-3 protease were incubated with 35S-Methionine-labeled CED-9 in the presence of 4 μl of C. elegans nuclei as in Fig. 3b, 4 μl of recombinant NPP-14 (125 nM final concentration) as in Fig. 4d, or 4 μl of the caspase inhibitor, iodoacetic acid (10 nM final concentration). Cleavage of the 31 kDa CED-9 protein by active CED-3 generated a 24 kDa cleavage product.

Supplementary Figure 3 Analysis of CED-3 autoactivation and CED-4-induced CED-3 activation in the presence of C. elegans nuclei.

(a) Repeat experiments of Fig. 3a. (b) Repeat experiments of Fig. 3b. (c) Repeat experiments of Fig. 3c. (d) Quantitative analysis of three independent CED-3 activation experiments without or with nuclei (shown in a-c and Fig. 3a-c, respectively). In each experiment, the combined intensity (Ia-t) of active CED-3 bands (17 kDa and 15 kDa bands very close together and indicated with red brackets) at the 25-min, 45-min, 60-min, or 90-min time point of each CED-3 protein (wild-type or mutant) and the intensity (Iz-25) of the CED-3 zymogen band at the 25-min time point were quantified using the Image J software (see Online Methods). The relative level of the CED-3 zymogen activation (Ca-t) at each time point (45-min, 60-min, or 90-min) in each experiments was determined as follow: Ca-t=(Ia-t-Ia-25)/Iz-25 (t indicates 45-min, 60-min, or 90-min time point). The average level of the CED-3 zymogen activation (Ca-t) at each time point was then determined from three independent experiments. Error bars are s.e.m. Compared with the corresponding Ca-t from wild-type CED-3, *** P < 0.001. ** P < 0.01. * P < 0.05 (two-sided t-tests).

Supplementary Figure 4 NPP-14 interacts with the CED-3 zymogen and its prodomain but not with the catalytic region of CED-3.

(a) Results of pull-down experiments between GST or GST-NPP-14 and the bacterial lysate containing 3.5 nM CED-3(A449V)-FLAG or CED-3(206-503, A449V)-FLAG (see Online Methods). The A449V mutation in CED-3 was used to block self-activation of the CED-3 zymogens in bacteria so that their potential interaction with NPP-14 can be examined. The left panel shows Coomassie Blue staining. The middle and right panels show immunoblotting using an anti-FLAG antibody. (b) An uncropped gel image of the one shown in Fig. 4a (lanes 10-14). (c) Cell corpse assays in 3-fold transgenic embryos of the indicated genotypes after heat-shock treatment (see Online Methods). Data shown are mean ± s.e.m (n=15). ** P < 0.01 (two-sided t-tests)

Supplementary Figure 5 Analysis of CED-3 autoactivation and CED-4-induced CED-3 activation in the presence of NPP-14.

(a) Repeat experiments of Fig. 4c. (b) Repeat experiments of Fig. 4d. (c) Repeat experiments of Fig. 4e. (d) Quantitative analysis of three independent CED-3 activation experiments without or with NPP-14 (shown in a-c and Fig. 3c-e, respectively). In each experiment, the combined intensity (Ia-t) of active CED-3 bands (17 kDa and 15 kDa bands very close together and indicated with red brackets) at the 25-min, 45-min, 60-min, or 90-min time point of each CED-3 protein (wild-type or mutant) and the intensity (Iz-25) of the CED-3 zymogen band at the 25-min time point were quantified using the Image J software (see Online Methods). The relative level of the CED-3 zymogen activation (Ca-t) at each time point (45-min, 60-min, or 90-min) in each experiments was determined as follow: Ca-t=(Ia-t-Ia-25)/Iz-25 (t indicates 45-min, 60-min, or 90-min time point). The average level of the CED-3 zymogen activation (Ca-t) at each time point was then determined from three independent experiments. Error bars are s.e.m. Compared with the corresponding Ca-t from wild-type CED-3, *** P < 0.001. ** P < 0.01. * P < 0.05 (two-sided t-tests).

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Chen, X., Wang, Y., Chen, YZ. et al. Regulation of CED-3 caspase localization and activation by C. elegans nuclear-membrane protein NPP-14. Nat Struct Mol Biol 23, 958–964 (2016). https://doi.org/10.1038/nsmb.3308

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