Analysis of Protein Processing by N-terminal Proteomics Reveals Novel Species-specific Substrate Determinants of Granzyme B Orthologs

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, l...

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Bibliographic Details
Published in:Molecular & cellular proteomics 2009-02, Vol.8 (2), p.258-272
Main Authors: Van Damme, Petra, Maurer-Stroh, Sebastian, Plasman, Kim, Van Durme, Joost, Colaert, Niklaas, Timmerman, Evy, De Bock, Pieter-Jan, Goethals, Marc, Rousseau, Frederic, Schymkowitz, Joost, Vandekerckhove, Joeül, Gevaert, Kris
Format: Article
Language:English
Subjects:
Amino Acid Motifs
Amino Acid Sequence
Animals
Apoptosis
Cell Death
Cell Line
Granzymes - chemistry
Granzymes - metabolism
Humans
Killer Cells, Natural - cytology
Mice
Models, Molecular
Molecular Sequence Data
Peptides - chemistry
Phylogeny
Protein Processing, Post-Translational
Proteome - chemistry
Proteomics - methods
Reproducibility of Results
Sequence Homology, Amino Acid
Species Specificity
Substrate Specificity
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Summary: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.
ISSN:1535-9476
1535-9484
DOI:10.1074/mcp.M800060-MCP200