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Title: Importance of extended protease substrate recognition motifs in steering BNIP-2 cleavage by human and mouse granzymes B
Authors: Van Damme, P
Plasman, K
Vandemoortele, G
Jonckheere, V
Maurer-Stroh, S 
Gevaert, K
Keywords: bnip2 protein
granzyme B
protein bcl 2
signal peptide
unclassified drug
BNIPL protein, human
peptide hydrolase
signal transducing adaptor protein
amino acid sequence
animal cell
cell death
cell lysate
controlled study
enzyme specificity
freeze thawing
human cell
in vitro study
jurkat cell line
liquid chromatography
nucleotide sequence
protein expression
protein function
protein motif
protein processing
protein protein interaction
signal transduction
site directed mutagenesis
tandem mass spectrometry
translation initiation
enzyme specificity
molecular genetics
sequence homology
Adaptor Proteins, Signal Transducing
Amino Acid Sequence
Chromatography, Liquid
Molecular Sequence Data
Peptide Hydrolases
Sequence Homology, Amino Acid
Substrate Specificity
Tandem Mass Spectrometry
Issue Date: 2014
Citation: Van Damme, P, Plasman, K, Vandemoortele, G, Jonckheere, V, Maurer-Stroh, S, Gevaert, K (2014). Importance of extended protease substrate recognition motifs in steering BNIP-2 cleavage by human and mouse granzymes B. BMC Biochemistry 15 (1) : 21. ScholarBank@NUS Repository.
Rights: Attribution 4.0 International
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 IEAD75 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 IEAD28. 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 I29 > 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. © 2014 Van Damme et al.; licensee BioMed Central Ltd.
Source Title: BMC Biochemistry
ISSN: 14712091
DOI: 10.1186/1471-2091-15-21
Rights: Attribution 4.0 International
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