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Sci Rep. 2017 Feb 23;7:43135. doi: 10.1038/srep43135.

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

Author information

1
Department of Bioorganic Chemistry, Faculty of Chemistry, Wroclaw University of Technology, Wyb. Wyspianskiego 27, 50-370 Wroclaw, Poland.
2
NCI-designated Cancer Center, Sanford-Burnham Prebys Medical Discovery Institute, La Jolla, CA 92037, USA.
3
Laboratory of Neurobiology, Nencki Institute of Experimental Biology, Polish Academy of Sciences, 02-093 Warsaw, Poland.
4
Kennedy Institute of Rheumatology, University of Oxford, United Kingdom.
5
Department of Biochemistry and Molecular and Structural Biology, Jožef Stefan Institute, SI-1000 Ljubljana, Slovenia.
6
Centre for Blood Research, Department of Oral Biological and Medical Sciences, University of British Columbia, Vancouver, BC V6T 1Z3, Canada.

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.

PMID:
28230157
PMCID:
PMC5322338
DOI:
10.1038/srep43135
[Indexed for MEDLINE]
Free PMC Article

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