title: Highly sensitive and adaptable fluorescence-quenched pair discloses the substrate specificity profiles in diverse protease families.
abstract: Internally quenched fluorescent (IQF) peptide substrates originating from FRET (Förster Resonance Energy Transfer) are powerful tool for examining the activity and specificity of proteases, and a variety of donor/acceptor pairs are extensively used to design individual substrates and combinatorial libraries. We developed a highly sensitive and adaptable donor/acceptor pair that can be used to investigate the substrate specificity of cysteine proteases, serine proteases and metalloproteinases. This novel pair comprises 7-amino-4-carbamoylmethylcoumarin (ACC) as the fluorophore and 2,4-dinitrophenyl-lysine (Lys(DNP)) as the quencher. Using caspase-3, caspase-7, caspase-8, neutrophil elastase, legumain, and two matrix metalloproteinases (MMP2 and MMP9), we demonstrated that substrates containing ACC/Lys(DNP) exhibit 7 to 10 times higher sensitivity than conventional 7-methoxy-coumarin-4-yl acetic acid (MCA)/Lys(DNP) substrates; thus, substantially lower amounts of substrate and enzyme can be used for each assay. We therefore propose that the ACC/Lys(DNP) pair can be considered a novel and sensitive scaffold for designing substrates for any group of endopeptidases. We further demonstrate that IQF substrates containing unnatural amino acids can be used to investigate protease activities/specificities for peptides containing post-translationally modified amino acids. Finally, we used IQF substrates to re-investigate the P1-Asp characteristic of caspases, thus demonstrating that some human caspases can also hydrolyze substrates after glutamic acid.
authors.affiliation: Department of Bioorganic Chemistry, Faculty of Chemistry, Wroclaw University of Technology, Wyb. Wyspianskiego 27, 50-370 Wroclaw, Poland.|NCI-designated Cancer Center, Sanford-Burnham Prebys Medical Discovery Institute, La Jolla, CA 92037, USA.
authors.forename: Marcin
authors.initials: M
authors.lastname: Poreba
authors.affiliation: Department of Bioorganic Chemistry, Faculty of Chemistry, Wroclaw University of Technology, Wyb. Wyspianskiego 27, 50-370 Wroclaw, Poland.
authors.forename: Aleksandra
authors.initials: A
authors.lastname: Szalek
authors.affiliation: Department of Bioorganic Chemistry, Faculty of Chemistry, Wroclaw University of Technology, Wyb. Wyspianskiego 27, 50-370 Wroclaw, Poland.
authors.forename: Wioletta
authors.initials: W
authors.lastname: Rut
authors.affiliation: Department of Bioorganic Chemistry, Faculty of Chemistry, Wroclaw University of Technology, Wyb. Wyspianskiego 27, 50-370 Wroclaw, Poland.|NCI-designated Cancer Center, Sanford-Burnham Prebys Medical Discovery Institute, La Jolla, CA 92037, USA.
authors.forename: Paulina
authors.initials: P
authors.lastname: Kasperkiewicz
authors.affiliation: Laboratory of Neurobiology, Nencki Institute of Experimental Biology, Polish Academy of Sciences, 02-093 Warsaw, Poland.
authors.forename: Izabela
authors.initials: I
authors.lastname: Rutkowska-Wlodarczyk
authors.affiliation: NCI-designated Cancer Center, Sanford-Burnham Prebys Medical Discovery Institute, La Jolla, CA 92037, USA.
authors.forename: Scott J
authors.initials: SJ
authors.lastname: Snipas
authors.affiliation: Kennedy Institute of Rheumatology, University of Oxford, United Kingdom.
authors.forename: Yoshifumi
authors.initials: Y
authors.lastname: Itoh
authors.affiliation: Department of Biochemistry and Molecular and Structural Biology, Jožef Stefan Institute, SI-1000 Ljubljana, Slovenia.
authors.forename: Dusan
authors.initials: D
authors.lastname: Turk
authors.affiliation: Department of Biochemistry and Molecular and Structural Biology, Jožef Stefan Institute, SI-1000 Ljubljana, Slovenia.
authors.forename: Boris
authors.initials: B
authors.lastname: Turk
authors.affiliation: Centre for Blood Research, Department of Oral Biological and Medical Sciences, University of British Columbia, Vancouver, BC V6T 1Z3, Canada.
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Overall
authors.affiliation: Laboratory of Neurobiology, Nencki Institute of Experimental Biology, Polish Academy of Sciences, 02-093 Warsaw, Poland.
authors.forename: Leszek
authors.initials: L
authors.lastname: Kaczmarek
authors.affiliation: NCI-designated Cancer Center, Sanford-Burnham Prebys Medical Discovery Institute, La Jolla, CA 92037, USA.
authors.forename: Guy S
authors.initials: GS
authors.lastname: Salvesen
authors.affiliation: Department of Bioorganic Chemistry, Faculty of Chemistry, Wroclaw University of Technology, Wyb. Wyspianskiego 27, 50-370 Wroclaw, Poland.
authors.forename: Marcin
authors.initials: M
authors.lastname: Drag
mesh_terms: D003374:Coumarins / Q000378:metabolism; D057056:Cysteine Proteases / Q000378:metabolism*; D005470:Fluorometry / Q000379:methods*; D006801:Humans; D008239:Lysine / Q000031:analogs & derivatives / Q000378:metabolism; D045726:Metalloproteases / Q000378:metabolism*; D012680:Sensitivity and Specificity; D057057:Serine Proteases / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: C411664:7-amino-4-carbamoylmethylcoumarin; D003374:Coumarins; C007493:epsilon-dinitrophenyllysine; D057056:Cysteine Proteases; D045726:Metalloproteases; D057057:Serine Proteases; D008239:Lysine