Development of the first internally-quenched fluorescent substrates of human cathepsin C: The application in the enzyme detection in biological samples

Arch Biochem Biophys. 2016 Dec 15;612:91-102. doi: 10.1016/j.abb.2016.10.007. Epub 2016 Oct 13.

Abstract

Cathepsin C is a widely expressed cysteine exopeptidase that is mostly recognized for the activation of the granule-associated proinflammatory serine proteases in neutrophils, cytotoxic T lymphocytes and mast cells. It has been shown that the enzyme can be secreted extracellularly; however, its occurrence in human bodily fluids/physiological samples has not been thoroughly studied. In the course of this study, the first fluorescence resonance energy transfer peptides for the measurement of the activity of human cathepsin C were designed and synthesized. Two series of tetra- and pentapeptide substrates enabled the detailed S' specificity study of cathepsin C, which has been examined for the first time. The extensive enzymatic studies of the obtained compounds resulted in the selection of the highly specific and selective substrate Thi-Ala(Mca)-Ser-Gly-Tyr(3-NO2)-NH2, which was successfully employed for the detection of cathepsin C activity in complex biological samples such as cell lysates, urine and bronchoalveolar lavage fluids. Molecular docking of the selected substrate was performed in order to better understand the binding mode of the substrates in the active site of cathepsin C.

Keywords: Cathepsin C; FRET substrates; Peptidomimetics; cysteine proteases.

MeSH terms

  • Catalytic Domain
  • Cathepsin C / chemistry*
  • Cathepsin L / chemistry
  • Fluorescence Resonance Energy Transfer
  • Humans
  • Hydrogen-Ion Concentration
  • Inflammation
  • Kinetics
  • Mast Cells / cytology
  • Microscopy, Fluorescence / methods*
  • Molecular Conformation
  • Molecular Docking Simulation
  • Neutrophils / metabolism
  • Peptides / chemistry
  • Protein Binding
  • Recombinant Proteins / chemistry
  • Substrate Specificity
  • T-Lymphocytes, Cytotoxic / cytology

Substances

  • Peptides
  • Recombinant Proteins
  • Cathepsin C
  • Cathepsin L