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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Main Authors: Van Damme, Petra, Plasman, Kim, Vandemoortele, Giel, Jonckheere, Veronique, Maurer-Stroh, Sebastian, Gevaert, Kris
Other Authors: School of Biological Sciences
Format: Article
Language:English
Published: 2014
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Online Access:https://hdl.handle.net/10356/102506
http://hdl.handle.net/10220/24274
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spelling 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
institution Nanyang Technological University
building NTU Library
continent Asia
country Singapore
Singapore
content_provider NTU Library
collection DR-NTU
language English
topic DRNTU::Science::Biological sciences::Biochemistry
spellingShingle 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
description 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.
author2 School of Biological Sciences
author_facet 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
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