Abstract
Caspases are a family of proteases found in all metazoans, including a dozen in humans, that drive the terminal stages of apoptosis as well as other cellular remodeling and inflammatory events. Caspases are named because they are cysteine class enzymes shown to cleave after aspartate residues. In the past decade, we and others have developed unbiased proteomic methods that collectively identified ~2000 native proteins cleaved during apoptosis after the signature aspartate residues. Here, we explore non-aspartate cleavage events and identify 100s of substrates cleaved after glutamate in both human and murine apoptotic samples. The extended consensus sequence patterns are virtually identical for the aspartate and glutamate cleavage sites suggesting they are cleaved by the same caspases. Detailed kinetic analyses of the dominant apoptotic executioner caspases-3 and -7 show that synthetic substrates containing DEVD↓ are cleaved only twofold faster than DEVE↓, which is well within the 500-fold range of rates that natural proteins are cut. X-ray crystallography studies confirm that the two acidic substrates bind in virtually the same way to either caspases-3 or -7 with minimal adjustments to accommodate the larger glutamate. Lastly, during apoptosis we found 121 proteins cleaved after serine residues that have been previously annotated to be phosphorylation sites. We found that caspase-3, but not caspase-7, can cleave peptides containing DEVpS↓ at only threefold slower rate than DEVD↓, but does not cleave the unphosphorylated serine peptide. There are only a handful of previously reported examples of proteins cleaved after glutamate and none after phosphorserine. Our studies reveal a much greater promiscuity for cleaving after acidic residues and the name 'cacidase' could aptly reflect this broader specificity.
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Abbreviations
- AFC:
-
7-amino-4-timethylfluoro-coumarin
- Ac:
-
acetyl
- Cmk:
-
chloromethylketone
- DNP:
-
dinitrophenol
- DTT:
-
dithiothreitol
- EDTA:
-
ethylenediaminetetraacetic acid
- HEPES:
-
2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid
- FBS:
-
fetal bovine serum
- Fmk:
-
fluormethylketone
- GO:
-
gene ontology
- RMSD:
-
root mean square deviation
- SDS:
-
sodium dodecyl sulfate
- PBS:
-
phosphate-buffered saline
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Acknowledgements
We thank Guy Salvesen and Scott Snipas at the Sanford Burnham Medical Research Institute for their helpful discussions and repeating some of kinetics with our AFC subtrates. We thank Emily Crawford, Zachary Hill (University of California, San Francisco), Dennis Wolan and Robyn Stanfield (The Scripps Research Institute) for helpful discussions and the staff of the ALS beamline 8.3.1 for support. This project was supported in part by Grants from National Institutes of Health R21 CA186077 (C.S.C.), R01 GM081051, R01 GM097316, and RO1 CA154802 (J.A.W.). It was also supported in part by NIH Training Grant T32 GM007175 (J.E.S.), the Global Healthy Living Foundation (J.E.S), NIH F31 CA180378 (T.J.R.) and the postdoctoral fellowships of a Banting Postdoctoral Fellowship from the Canadian Institutes of Health Research, Government of Canada (O.J.) and the Damon Runyon Cancer Research Foundation 2082-11 (N.D.T.).
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Seaman, J., Julien, O., Lee, P. et al. Cacidases: caspases can cleave after aspartate, glutamate and phosphoserine residues. Cell Death Differ 23, 1717–1726 (2016). https://doi.org/10.1038/cdd.2016.62
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DOI: https://doi.org/10.1038/cdd.2016.62
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