@article{5786306,
  abstract     = {{Background: Previous screening of the substrate repertoires and substrate specificity profiles of granzymes resulted in long substrate lists highly likely containing bystander substrates. Here, a recently developed degradomics technology that allows distinguishing efficiently from less efficiently cleaved substrates was applied to study the degradome of mouse granzyme B (mGrB). 
Results: In vitro kinetic degradome analysis resulted in the identification of 37 mGrB cleavage events, 9 of which could be assigned as efficiently targeted ones. Previously, cleavage at the IEAD(75) tetrapeptide motif of Bid was shown to be efficiently and exclusively targeted by human granzyme B (hGrB) and thus not by mGrB. Strikingly, and despite holding an identical P4-P1 human Bid (hBid) cleavage motif, mGrB was shown to efficiently cleave the BCL2/adenovirus E1B 19 kDa protein-interacting protein 2 or BNIP-2 at IEAD(28). Like Bid, BNIP-2 represents a pro-apoptotic Bcl-2 protein family member and a potential regulator of GrB induced cell death. Next, in vitro analyses demonstrated the increased efficiency of human and mouse BNIP-2 cleavage by mGrB as compared to hGrB indicative for differing Bid/BNIP-2 substrate traits beyond the P4-P1 IEAD cleavage motif influencing cleavage efficiency. Murinisation of differential primed site residues in hBNIP-2 revealed that, although all contributing, a single mutation at the P3' position was found to significantly increase the mGrB/hGrB cleavage ratio, whereas mutating the P1' position from I-29 > T yielded a 4-fold increase in mGrB cleavage efficiency. Finally, mutagenesis analyses revealed the composite BNIP 2 precursor patterns to be the result of alternative translation initiation at near-cognate start sites within the 5' leader sequence (5'UTR) of BNIP-2. 
Conclusions: Despite their high sequence similarity, and previously explained by their distinct tetrapeptide specificities observed, the substrate repertoires of mouse and human granzymes B only partially overlap. Here, we show that the substrate sequence context beyond the P4-P1 positions can influence orthologous granzyme B cleavage efficiencies to an unmatched extent. More specifically, in BNIP-2, the identical and hGrB optimal IEAD tetrapeptide substrate motif is targeted highly efficiently by mGrB, while this tetrapeptide motif is refractory towards mGrB cleavage in Bid.}},
  articleno    = {{21}},
  author       = {{Van Damme, Petra and Plasman, Kim and Vandemoortele, Giel and Jonckheere, Veronique and Maurer-Stroh, Sebastian and Gevaert, Kris}},
  issn         = {{1471-2091}},
  journal      = {{BMC BIOCHEMISTRY}},
  keywords     = {{N-terminal COFRADIC,Degradomics,Near-cognate translation initiation,FRACTIONAL DIAGONAL CHROMATOGRAPHY,CASPASE ACTIVATION,CELL-DEATH,Extended substrate specificity,Granzyme B,Bid,BNIP-2,PROTEOMICS,CDC42GAP,PROTEINS,N-TERMINAL PEPTIDES,GENE-EXPRESSION,HOMOLOGY BCH DOMAIN,INDUCED APOPTOSIS}},
  language     = {{eng}},
  pages        = {{17}},
  title        = {{Importance of extended protease substrate recognition motifs in steering BNIP-2 cleavage by human and mouse granzymes B}},
  url          = {{http://dx.doi.org/10.1186/1471-2091-15-21}},
  volume       = {{15}},
  year         = {{2014}},
}

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