Importance of extended protease substrate recognition motifs in steering BNIP-2 cleavage by human and mouse granzymes B
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 cl...
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sg-ntu-dr.10356-1025062023-02-28T17:05:11Z Importance of extended protease substrate recognition motifs in steering BNIP-2 cleavage by human and mouse granzymes B Van Damme, Petra Plasman, Kim Vandemoortele, Giel Jonckheere, Veronique Maurer-Stroh, Sebastian Gevaert, Kris School of Biological Sciences DRNTU::Science::Biological sciences::Biochemistry 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. Published version 2014-12-01T08:10:12Z 2019-12-06T20:56:04Z 2014-12-01T08:10:12Z 2019-12-06T20:56:04Z 2014 2014 Journal Article 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-. 1471-2091 https://hdl.handle.net/10356/102506 http://hdl.handle.net/10220/24274 10.1186/1471-2091-15-21 25208769 en BMC biochemistry © 2014 Van Damme et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. 17 p. application/pdf |
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DRNTU::Science::Biological sciences::Biochemistry Van Damme, Petra Plasman, Kim Vandemoortele, Giel Jonckheere, Veronique Maurer-Stroh, Sebastian Gevaert, Kris Importance of extended protease substrate recognition motifs in steering BNIP-2 cleavage by human and mouse granzymes B |
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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. |
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School of Biological Sciences |
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School of Biological Sciences Van Damme, Petra Plasman, Kim Vandemoortele, Giel Jonckheere, Veronique Maurer-Stroh, Sebastian Gevaert, Kris |
format |
Article |
author |
Van Damme, Petra Plasman, Kim Vandemoortele, Giel Jonckheere, Veronique Maurer-Stroh, Sebastian Gevaert, Kris |
author_sort |
Van Damme, Petra |
title |
Importance of extended protease substrate recognition motifs in steering BNIP-2 cleavage by human and mouse granzymes B |
title_short |
Importance of extended protease substrate recognition motifs in steering BNIP-2 cleavage by human and mouse granzymes B |
title_full |
Importance of extended protease substrate recognition motifs in steering BNIP-2 cleavage by human and mouse granzymes B |
title_fullStr |
Importance of extended protease substrate recognition motifs in steering BNIP-2 cleavage by human and mouse granzymes B |
title_full_unstemmed |
Importance of extended protease substrate recognition motifs in steering BNIP-2 cleavage by human and mouse granzymes B |
title_sort |
importance of extended protease substrate recognition motifs in steering bnip-2 cleavage by human and mouse granzymes b |
publishDate |
2014 |
url |
https://hdl.handle.net/10356/102506 http://hdl.handle.net/10220/24274 |
_version_ |
1759856025721110528 |