title: Sensitive Versatile Fluorogenic Transmembrane Peptide Substrates for Rhomboid Intramembrane Proteases.
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 modifiablein vitroactivity 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.
authors.affiliation: From the Institute of Organic Chemistry and Biochemistry of the Czech Academy of Science, Flemingovo n. 2, Prague 166 10.|the First Faculty of Medicine, Charles University, Kateřinská 32, Prague 121 08, and.
authors.forename: Anežka
authors.initials: A
authors.lastname: Tichá
authors.affiliation: From the Institute of Organic Chemistry and Biochemistry of the Czech Academy of Science, Flemingovo n. 2, Prague 166 10.
authors.forename: Stancho
authors.initials: S
authors.lastname: Stanchev
authors.affiliation: From the Institute of Organic Chemistry and Biochemistry of the Czech Academy of Science, Flemingovo n. 2, Prague 166 10.|the Department of Biochemistry, Faculty of Science, Charles University, Hlavova 2030/8, Prague 128 43.
authors.forename: Jan
authors.initials: J
authors.lastname: Škerle
authors.affiliation: From the Institute of Organic Chemistry and Biochemistry of the Czech Academy of Science, Flemingovo n. 2, Prague 166 10.|the Department of Genetics and Microbiology, Faculty of Science, Charles University, Viničná 5, Prague 128 44.
authors.forename: Jakub
authors.initials: J
authors.lastname: Began
authors.affiliation: the Department of Biochemistry, Faculty of Science, Charles University, Hlavova 2030/8, Prague 128 43.|the Department of Physics and Materials Engineering, Tomas Bata University in Zlín, Faculty of Technology, nám. T.G. Masaryka 5555, 76001, Zlín, Czech Republic.
authors.forename: Marek
authors.initials: M
authors.lastname: Ingr
authors.affiliation: From the Institute of Organic Chemistry and Biochemistry of the Czech Academy of Science, Flemingovo n. 2, Prague 166 10.
authors.forename: Kateřina
authors.initials: K
authors.lastname: Švehlová
authors.affiliation: From the Institute of Organic Chemistry and Biochemistry of the Czech Academy of Science, Flemingovo n. 2, Prague 166 10.|the Department of Biochemistry, Faculty of Science, Charles University, Hlavova 2030/8, Prague 128 43.
authors.forename: Lucie
authors.initials: L
authors.lastname: Polovinkin
authors.affiliation: From the Institute of Organic Chemistry and Biochemistry of the Czech Academy of Science, Flemingovo n. 2, Prague 166 10.|the Department of Biochemistry, Faculty of Science, Charles University, Hlavova 2030/8, Prague 128 43.
authors.forename: Martin
authors.initials: M
authors.lastname: Růžička
authors.affiliation: From the Institute of Organic Chemistry and Biochemistry of the Czech Academy of Science, Flemingovo n. 2, Prague 166 10.
authors.forename: Lucie
authors.initials: L
authors.lastname: Bednárová
authors.affiliation: From the Institute of Organic Chemistry and Biochemistry of the Czech Academy of Science, Flemingovo n. 2, Prague 166 10.
authors.forename: Romana
authors.initials: R
authors.lastname: Hadravová
authors.affiliation: From the Institute of Organic Chemistry and Biochemistry of the Czech Academy of Science, Flemingovo n. 2, Prague 166 10.
authors.forename: Edita
authors.initials: E
authors.lastname: Poláchová
authors.affiliation: From the Institute of Organic Chemistry and Biochemistry of the Czech Academy of Science, Flemingovo n. 2, Prague 166 10.
authors.forename: Petra
authors.initials: P
authors.lastname: Rampírová
authors.affiliation: From the Institute of Organic Chemistry and Biochemistry of the Czech Academy of Science, Flemingovo n. 2, Prague 166 10.
authors.forename: Jana
authors.initials: J
authors.lastname: Březinová
authors.affiliation: From the Institute of Organic Chemistry and Biochemistry of the Czech Academy of Science, Flemingovo n. 2, Prague 166 10.
authors.forename: Václav
authors.initials: V
authors.lastname: Kašička
authors.affiliation: From the Institute of Organic Chemistry and Biochemistry of the Czech Academy of Science, Flemingovo n. 2, Prague 166 10.
authors.forename: Pavel
authors.initials: P
authors.lastname: Majer
authors.affiliation: From the Institute of Organic Chemistry and Biochemistry of the Czech Academy of Science, Flemingovo n. 2, Prague 166 10, kvido.strisovsky@uochb.cas.cz.
authors.forename: Kvido
authors.initials: K
authors.lastname: Strisovsky
mesh_terms: D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry*; D007700:Kinetics; D008081:Liposomes; D010447:Peptide Hydrolases / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D005456:Fluorescent Dyes; D008081:Liposomes; D010455:Peptides; D010447:Peptide Hydrolases
keywords: enzyme kinetics; enzyme mechanism; fluorescence resonance energy transfer (FRET); intramembrane proteolysis; membrane reconstitution; rhomboid protease; substrate specificity; transmembrane domain