Abstract
Caspases, cysteine proteases with aspartate specificity, are key players in programmed cell death across the metazoan lineage. Hundreds of apoptotic caspase substrates have been identified in human cells. Some have been extensively characterized, revealing key functional nodes for apoptosis signaling and important drug targets in cancer. But the functional significance of most cuts remains mysterious. We set out to better understand the importance of caspase cleavage specificity in apoptosis by asking which cleavage events are conserved across metazoan model species. Using N-terminal labeling followed by mass spectrometry, we identified 257 caspase cleavage sites in mouse, 130 in Drosophila, and 50 in Caenorhabditis elegans. The large majority of the caspase cut sites identified in mouse proteins were found conserved in human orthologs. However, while many of the same proteins targeted in the more distantly related species were cleaved in human orthologs, the exact sites were often different. Furthermore, similar functional pathways are targeted by caspases in all four species. Our data suggest a model for the evolution of apoptotic caspase specificity that highlights the hierarchical importance of functional pathways over specific proteins, and proteins over their specific cleavage site motifs.
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Abbreviations
- ppm:
-
part per million
- GST:
-
glutathione S-transferase
- GO:
-
gene ontology
- IPA:
-
Ingenuity Pathway Analysis
- SDS:
-
sodium dodecyl sulfate
- FBS:
-
fetal bovine serum
- eIF:
-
eukaryotic initiation factor
- RPL:
-
ribosomal protein, large subunit
- PBS:
-
phosphate buffered saline
- MEF:
-
mouse embryonic fibroblast
- PTM:
-
post-translational modification
- PMSF:
-
phenylmethanesulfonylfluoride
- AEBSF:
-
4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride
- EDTA:
-
Ethylenediaminetetraacetic acid
- IAM:
-
iodoacetamide
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Acknowledgements
We thank Sami Mahrus, David Wildes, Nicholas Agard, Hikari Yoshihara, Huy Nguyen, Gerald Hsu, and Kazutaka Shimbo for contributing to the human caspase substrate data set. We also thank Cynthia Kenyon and Aimee Kao for assistance with the C. elegans work, and David Maltby, Jonathan Trinidad, and Shenheng Guan and the rest of the UCSF Mass Spectrometry Facility, plus Iman Mohtashemi and Yan Chen of Thermo Scientific, for mass spectrometry assistance. We thank Bent Petersen for assistance with the NetSurfP server. This project was supported by a grant from the UCSF Stephen and Nancy Grand Multiple Myeloma Translational Initiative. It was also supported by the National Science Foundation GRFP (EDC), NIH Training Grant T32 GM007175 (JES and PCB), the Pharmaceutical Research and Manufacturers of America (AEB), National Institutes of Health R01 GM60595 (PCB), and National Institutes of Health R01 GM081051 (JAW). Mass spectrometry was performed at the Bio-Organic Biomedical Mass Spectrometry Resource at UCSF (ALB, Director), which is supported by grants from the National Center for Research Resources (5P41RR001614) and the National Institute of General Medical Sciences (8 P41 GM103481) from the National Institutes of Health.
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Crawford, E., Seaman, J., Barber, A. et al. Conservation of caspase substrates across metazoans suggests hierarchical importance of signaling pathways over specific targets and cleavage site motifs in apoptosis. Cell Death Differ 19, 2040–2048 (2012). https://doi.org/10.1038/cdd.2012.99
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DOI: https://doi.org/10.1038/cdd.2012.99
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