Abstract
E3 ubiquitin ligases target a growing number of pro- and anti-apoptotic proteins, including tumour suppressor p53, caspases, and the Bcl-2 family. The core apoptosis pathway is well conserved between mammals and Caenorhabditis elegans, but the extent to which ubiquitin ligases regulate apoptotic cell death is not known. To investigate the role of E3 ligases in apoptosis, we inhibited 108 of the 165 predicted E3 ubiquitin ligase genes by RNA interference and quantified apoptosis in the C. elegans germline after genotoxic stress. From this screen, we identified the homologous to E6-associated protein C terminus-domain E3 ligase EEL-1 as a positive regulator of apoptosis. Intriguingly, the human homologue of EEL-1, Huwe1/ARF-BP1/Mule/HectH9, has been reported to possess both pro- and anti-apoptotic functions through its ability to stimulate Mcl-1 and p53 degradation, respectively. Here, we demonstrate that eel-1 is required to promote DNA damage-induced germ cell apoptosis, but does not have a role in physiological germ cell apoptosis or developmental apoptosis in somatic tissue. Furthermore, eel-1 acts in parallel to the p53-like gene cep-1 and intersects the core apoptosis pathway upstream of the Bcl-2/Mcl-1 orthologue ced-9. Although ee1-1 mutants exhibit hypersensitivity to genotoxic stress they do not appear to be defective in DNA repair, suggesting a distinct role for EEL-1 in promoting damage-induced apoptosis in the germline.
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Abbreviations
- HECT:
-
homologous to E6-associated protein C terminus
- RING:
-
really interesting new gene
- PCD:
-
programmed cell death
- Mdm2:
-
murine double minute clone 2 oncoprotein
- IAP:
-
inhibitor of apoptosis protein
- SCF:
-
Skp-Cullin-F-box
- IR:
-
ionizing radiation
- NGM:
-
nematode growth media
- RNAi:
-
RNA interference
- HU:
-
hydroxyurea
- IPTG:
-
isopropyl-β-D-thiogalactopyranoside
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Acknowledgements
We thank the Caenorhabditis Genetics Center, which is funded by the National Institutes of Health Center for Research Resources for providing worm strains. We kindly thank H.R Horvitz (Massachusetts Institute of Technology) for providing the ced-9(n2812)/qC1 strain, B. Conradt and Stephane Rolland (Dartmouth University) for CED-9 antibody and assistance with the western blot protocol. This work was supported by a Canadian Institutes of Health Research award to W.B.D., Natural Sciences and Engineering Research Council of Canada Graduate Scholarship to A.R., and a Canadian Institutes of Health Strategic Training Fellow in the Excellence in Radiation Research for the 21st Century Program to M.L. We are grateful to A. Perrin and S. Ito for helpful input on the manuscript.
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Ross, A., Li, M., Yu, B. et al. The EEL-1 ubiquitin ligase promotes DNA damage-induced germ cell apoptosis in C. elegans. Cell Death Differ 18, 1140–1149 (2011). https://doi.org/10.1038/cdd.2010.180
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DOI: https://doi.org/10.1038/cdd.2010.180
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