Highly sensitive and adaptable fluorescence-quenched pair discloses the substrate specificity profiles in diverse protease families.
In: Scientific Reports, 2017-02-24, S. 43135-43135
Online
academicJournal
Zugriff:
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. [ABSTRACT FROM AUTHOR]
Titel: |
Highly sensitive and adaptable fluorescence-quenched pair discloses the substrate specificity profiles in diverse protease families.
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Autor/in / Beteiligte Person: | 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 |
Link: | |
Zeitschrift: | Scientific Reports, 2017-02-24, S. 43135-43135 |
Veröffentlichung: | 2017 |
Medientyp: | academicJournal |
ISSN: | 2045-2322 (print) |
DOI: | 10.1038/srep43135 |
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