Open AccessHighly sensitive and adaptable fluorescence-quenched pair discloses the substrate specificity profiles in diverse protease families
Author(s) -
Marcin Poręba,
Aleksandra Szalek,
Wioletta Rut,
Paulina Kasperkiewicz,
Izabela Rutkowska-Włodarczyk,
Scott J. Snipas,
Yoshifumi Itoh,
Dušan Turk,
Boris Turk,
Christopher M. Overall,
Leszek Kaczmarek,
Guy S. Salvesen,
Marcin Drąg
Publication year - 2017
Publication title -
scientific reports
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.24
H-Index - 213
ISSN - 2045-2322
DOI - 10.1038/srep43135
Subject(s) - proteases , amino acid , förster resonance energy transfer , protease , fluorophore , chemistry , caspase , lysine , substrate (aquarium) , cysteine , biochemistry , serine , matrix metalloproteinase , enzyme , fluorescence , biology , apoptosis , ecology , physics , quantum mechanics , programmed cell death
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.