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, 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.
Department of Medical Protein Research, Flanders Institute for Biotechnology (VIB), Ghent, Belgium.
Petra
P
Van Damme
Sebastian
S
Maurer-Stroh
Kim
K
Plasman
Joost
J
Van Durme
Niklaas
N
Colaert
Evy
E
Timmerman
Pieter-Jan
PJ
De Bock
Marc
M
Goethals
Frederic
F
Rousseau
Joost
J
Schymkowitz
Joël
J
Vandekerckhove
Kris
K
Gevaert
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D016923:Cell Death; D002460:Cell Line; D053804:Granzymes / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007694:Killer Cells, Natural / Q000166:cytology; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D010802:Phylogeny; D011499:Protein Processing, Post-Translational; D020543:Proteome / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013379:Substrate Specificity
D010455:Peptides; D020543:Proteome; D053804:Granzymes