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Sensitive versatile fluorogenic transmembrane peptide substrates for rhomboid intramembrane proteases

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dc.title Sensitive versatile fluorogenic transmembrane peptide substrates for rhomboid intramembrane proteases en
dc.contributor.author Tichá, Anežka
dc.contributor.author Stanchev, Stancho
dc.contributor.author Škerle, Jan
dc.contributor.author Began, Jakub
dc.contributor.author Ingr, Marek
dc.contributor.author Švehlová, Kateřina
dc.contributor.author Polovinkin, Lucie
dc.contributor.author Růžička, Martin
dc.contributor.author Bednářová, Lucie
dc.contributor.author Hadravová, Romana
dc.contributor.author Poláchová, Edita
dc.contributor.author Rampírová, Petra
dc.contributor.author Březinová, Jana
dc.contributor.author Kašička, Václav
dc.contributor.author Majer, Pavel
dc.contributor.author Stříšovský, Kvido
dc.relation.ispartof Journal of Biological Chemistry
dc.identifier.issn 0021-9258 Scopus Sources, Sherpa/RoMEO, JCR
dc.date.issued 2017
utb.relation.volume 292
utb.relation.issue 7
dc.citation.spage 2703
dc.citation.epage 2713
dc.type article
dc.language.iso en
dc.publisher American Society for Biochemistry and Molecular Biology Inc.
dc.identifier.doi 10.1074/jbc.M116.762849
dc.relation.uri http://www.jbc.org/content/early/2017/01/09/jbc.M116.762849
dc.description.abstract Rhomboid proteases are increasingly being explored as potential drug targets, but their potent and specific inhibitors are not available, and strategies for inhibitor development are hampered by the lack of widely usable and easily modifiable in vitro activity assays. Here we address this bottleneck and report on the development of new fluorogenic transmembrane peptide substrates, which are cleaved by several unrelated rhomboid proteases, can be used both in detergent micelles and in liposomes, and contain red-shifted fluorophores that are suitable for high-throughput screening of compound libraries. We show that nearly the entire transmembrane domain of the substrate is important for efficient cleavage, implying that it extensively interacts with the enzyme. Importantly, we demonstrate that in the detergent micelle system, commonly used for the enzymatic analyses of intramembrane proteolysis, the cleavage rate strongly depends on detergent concentration, because the reaction proceeds only in the micelles. Furthermore, we show that the catalytic efficiency and selectivity toward a rhomboid substrate can be dramatically improved by targeted modification of the sequence of its P5 to P1 region. The fluorogenic substrates that we describe and their sequence variants should find wide use in the detection of activity and development of inhibitors of rhomboid proteases. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc. en
utb.faculty Faculty of Technology
dc.identifier.uri http://hdl.handle.net/10563/1006900
utb.identifier.obdid 43877119
utb.identifier.scopus 2-s2.0-85013219026
utb.identifier.wok 000395535100013
utb.identifier.pubmed 28069810
utb.identifier.coden JBCHA
utb.source j-scopus
dc.date.accessioned 2017-06-27T08:13:11Z
dc.date.available 2017-06-27T08:13:11Z
dc.description.sponsorship 15-01948S, GA ČR, Grantová Agentura České Republiky; 2329, EMBO, European Molecular Biology Organization; MOE, Ministry of Education; 232313, Univerzita Karlova v Praze; P208-12-G016, GA ČR, Grantová Agentura České Republiky
dc.description.sponsorship EMBO Installation Grant [2329]; Ministry of Education, Youth and Sports of the Czech Republic Projects [LK11206, LO1302]; National Subvention for Development of Research Organizations [RVO: 61388963]; Marie Curie Career Integration Grant [304154]
utb.contributor.internalauthor Ingr, Marek
utb.fulltext.affiliation Anežka Tichá 1,4,# , Stancho Stanchev 1,# , Jan Škerle 1,2 , Jakub Began 1,3 , Marek Ingr 2,5 , Kateřina Švehlová 1 , Lucie Polovinkin 1,2,$ , Martin Růžička 1,2 , Lucie Bednárová 1 , Romana Hadravová 1 , Edita Poláchová 1 , Petra Rampírová 1 , Jana Březinová 1 , Václav Kašička 1 , Pavel Majer 1 and Kvido Strisovsky 1 * 1 The Institute of Organic Chemistry and Biochemistry of the Czech Academy of Science, Flemingovo n. 2, Prague, 166 10, Czech Republic 2 Department of Biochemistry, Faculty of Science, Charles University, Hlavova 2030/8, Prague, 128 43, Czech Republic 3 Department of Genetics and Microbiology, Faculty of Science, Charles University, Viničná 5, Prague, 128 44, Czech Republic 4 First Faculty of Medicine, Charles University, Kateřinská 32, Prague, 121 08, Czech Republic 5 Tomas Bata University in Zlín, Faculty of Technology, Department of Physics and Materials Engineering, nám. T.G. Masaryka 5555, 76001, Zlín, Czech Republic $ Present address: Institut de Biologie Structurale, 71 avenue des Martyrs, Grenoble, 38044, France * corresponding author: kvido.strisovsky@uochb.cas.cz, Institute of Organic Chemistry and Biochemistry, Flemingovo n. 2, Prague, 166 10, Czech Republic, tel.: +420 220 183 468 # equal contribution
utb.fulltext.dates J. Biol. Chem. published online January 9, 2017
utb.fulltext.sponsorship We thank Steven Verhelst (University of Leuven, Belgium) for his kind gift of isocoumarin S037, Matthew Freeman (Oxford University, United Kingdom) for his kind gift of the inhibitor L42, Zdeněk Voburka and Radko Souček for amino acid analyses, Mirka Blechová for peptide synthesis and purification, and Blanka Collis for critical reading of the manuscript. KS was a recipient of the Purkyne Fellowship of the Academy of Sciences of the Czech Republic and acknowledges support also from EMBO (Installation Grant no. 2329), Ministry of Education, Youth and Sports of the Czech Republic (projects no. LK11206 and LO1302), Marie Curie Career Integration Grant (project no. 304154), and the National Subvention for Development of Research Organizations (RVO: 61388963) to the Institute of Organic Chemistry and Biochemistry. JS and JB were supported by PhD grant projects no. 232313 and 170214, respectively, from the Grant Agency of Charles University (GA UK) in Prague. MI has been supported by the Czech Science Foundation, grant no. P208-12-G016 (Center of Excellence), and VK and MR were supported by the Czech Science Foundation, grant no. 15-01948S.
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