Highly sensitive and adaptable fluorescence-quenched pair discloses the substrate specificity profiles in diverse protease families

• Published in: Scientific reports Vol. 7; no. 1; p. 43135
• Main Authors: Poreba, Marcin, Szalek, Aleksandra, Rut, Wioletta, Kasperkiewicz, Paulina, Rutkowska-Wlodarczyk, Izabela, Snipas, Scott J., Itoh, Yoshifumi, Turk, Dusan, Turk, Boris, Overall, Christopher M., Kaczmarek, Leszek, Salvesen, Guy S., Drag, Marcin
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 23.02.2017; Nature Publishing Group
• Subjects: 631/45/468; 631/92/2132; 631/92/96; Acetic acid; Amino acids; Caspase; Caspase-3; Caspase-7; Caspase-8; Combinatorial libraries; Coumarin; Coumarins - metabolism; Cysteine; Cysteine Proteases - metabolism; Elastase; Fluorescence resonance energy transfer; Fluorometry - methods; Gelatinase A; Gelatinase B; Glutamic acid; Humanities and Social Sciences; Humans; Legumain; Lysine; Lysine - analogs & derivatives; Lysine - metabolism; Metalloproteases - metabolism; multidisciplinary; Post-translation; Proteinase; Science; Sensitivity and Specificity; Serine; Serine Proteases - metabolism; Substrate Specificity
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/srep43135

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.