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

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

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Coumarins / metabolism
  • Cysteine Proteases / metabolism*
  • Fluorometry / methods*
  • Humans
  • Lysine / analogs & derivatives
  • Lysine / metabolism
  • Metalloproteases / metabolism*
  • Sensitivity and Specificity
  • Serine Proteases / metabolism*
  • Substrate Specificity*

Substances

  • 7-amino-4-carbamoylmethylcoumarin
  • Coumarins
  • epsilon-dinitrophenyllysine
  • Cysteine Proteases
  • Metalloproteases
  • Serine Proteases
  • Lysine