Journal
MOLECULAR & CELLULAR PROTEOMICS
Volume 8, Issue 2, Pages 258-272Publisher
ELSEVIER
DOI: 10.1074/mcp.M800060-MCP200
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Funding
- Fund for Scientific Research-Flanders (Belgium) [G.0156.05, G.0077.06, G.0042.07]
- Ghent University [BOF07/GOA/012]
- Inter University Attraction Poles [IUAP06]
- European Union Interaction Proteome
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Using a targeted peptide-centric proteomics approach, we performed in vitro protease substrate profiling of the apoptotic serine protease granzyme B resulting in the delineation of more than 800 cleavage sites in 322 human and 282 mouse substrates, encompassing the known substrates Bid, caspase-7, lupus La protein, and fibrillarin. Triple SILAC (stable isotope labeling by amino acids in cell culture) further permitted intra-experimental evaluation of species-specific variations in substrate selection by the mouse or human granzyme B ortholog. For the first time granzyme B substrate specificities were directly mapped on a proteomic scale and revealed unknown cleavage specificities, uncharacterized extended specificity profiles, and macromolecular determinants in substrate selection that were confirmed by molecular modeling. We further tackled a substrate hunt in an in vivo setup of natural killer cell-mediated cell death confirming in vitro characterized granzyme B cleavages next to several other unique and hitherto unreported proteolytic events in target cells. Molecular & Cellular Proteomics 8:258-272, 2009.
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