Abstract
The mammalian cell death network comprises three distinct functional modules: apoptosis, autophagy and programmed necrosis. Currently, the field lacks systems level approaches to assess the extent to which the intermodular connectivity affects cell death performance. Here, we developed a platform that is based on single and double sets of RNAi-mediated perturbations targeting combinations of apoptotic and autophagic genes. The outcome of perturbations is measured both at the level of the overall cell death responses, using an unbiased quantitative reporter, and by assessing the molecular responses within the different functional modules. Epistatic analyses determine whether seemingly unrelated pairs of proteins are genetically linked. The initial running of this platform in etoposide-treated cells, using a few single and double perturbations, identified several levels of connectivity between apoptosis and autophagy. The knock down of caspase3 turned on a switch toward autophagic cell death, which requires Atg5 or Beclin-1. In addition, a reciprocal connection between these two autophagic genes and apoptosis was identified. By applying computational tools that are based on mining the protein–protein interaction database, a novel biochemical pathway connecting between Atg5 and caspase3 is suggested. Scaling up this platform into hundreds of perturbations potentially has a wide, general scope of applicability, and will provide the basis for future modeling of the cell death network.
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Abbreviations
- PCD:
-
programmed cell death
- PPI:
-
protein–protein interaction
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Acknowledgements
We thank Helena Sabanay for her assistance in performing the TEM experiments, Sara Barak for performing the antiviral response assay, and Yulia Idelchuk for essential technical help. We thank Yitzhak Pilpel for fruitful discussions and for commenting on the manuscript, Edna Schechtman for help and advice in statistical analysis and Z Kam for the help in setting up the live imaging system. We thank N Mizushima and T Yoshimori for providing the GFP-LC3 plasmid, YK Jung for the Atg5-HA plasmid, ME Peter for the Flag-DEDD plasmid and Y Lazebnik for providing anti-Caspase9 and anti-Caspase7 antibodies. This work was supported by the Kahn Fund for System Biology at the Weizmann Institute of Science and by the EU grant (APO-SYS) to A Kimchi. A Kimchi is the incumbent of the Helena Rubinstein Chair of Cancer Research. R Sharan and E Ruppin were supported by a converging technologies grant from the Israel Science Foundation.
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Zalckvar, E., Yosef, N., Reef, S. et al. A systems level strategy for analyzing the cell death network: implication in exploring the apoptosis/autophagy connection. Cell Death Differ 17, 1244–1253 (2010). https://doi.org/10.1038/cdd.2010.7
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DOI: https://doi.org/10.1038/cdd.2010.7
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