title: A targeted protease substrate for a quantitative determination of protease activities in the endolysosomal pathway.
abstract: Inside the cell, proteases act in concert in the degradation of proteins and peptides. In order to understand the significance of an individual proteolytic activity within an ensemble of proteases, protocols and probes are required that enable a quantitative determination of the contribution of a protease to the break-down of a given substrate. Here we present a fluorescence resonance energy transfer-based probe and protocols for a quantitative determination of proteolytic activities inside the endolysosomal compartment. A peptide substrate that is readily cleaved by different cathepsins is flanked by fluorescein and tetramethylrhodamine-labeled lysine residues. Efficient endolysosomal targeting of the substrate is achieved by N-terminal elongation with the cell-penetrating peptide nona-arginine. The proteasome inhibitor lactacystin has a small, but significant effect on the break-down of the substrate, thus demonstrating that only a minor fraction of the peptide reaches the cytoplasm in its intact form. Nona-arginine therefore constitutes a highly efficient low-molecular-weight moiety for targeting the endolysosomal compartment.
authors.affiliation: Eberhard Karls University Tübingen, Department of Molecular Biology, Interfaculty Institute for Cell Biology, Auf der Morgenstelle 15, 72076 Tübingen, Germany.
authors.forename: Rainer
authors.initials: R
authors.lastname: Fischer
authors.forename: Daniel
authors.initials: D
authors.lastname: Bächle
authors.forename: Mariola
authors.initials: M
authors.lastname: Fotin-Mleczek
authors.forename: Günther
authors.initials: G
authors.lastname: Jung
authors.forename: Hubert
authors.initials: H
authors.lastname: Kalbacher
authors.forename: Roland
authors.initials: R
authors.lastname: Brock
mesh_terms: D002384:Catalysis / Q000187:drug effects; D002401:Cathepsin B / Q000378:metabolism; D002402:Cathepsin D / Q000378:metabolism; D056668:Cathepsin L; D002403:Cathepsins / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000378:metabolism; D004705:Endocytosis / Q000187:drug effects; D011992:Endosomes / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005452:Fluoresceins / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D008247:Lysosomes / Q000201:enzymology*; D009842:Oligopeptides / Q000737:chemistry; D010436:Pepstatins / Q000494:pharmacology; D010447:Peptide Hydrolases / Q000378:metabolism*; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology; D012235:Rhodamines / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013347:Subcellular Fractions / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: C114557:5-carboxytetramethylrhodamine succinimidyl ester; D004791:Enzyme Inhibitors; D005452:Fluoresceins; D005456:Fluorescent Dyes; D009842:Oligopeptides; D010436:Pepstatins; D010455:Peptides; D011480:Protease Inhibitors; D012235:Rhodamines; C491427:nonaarginine; C024098:6-carboxyfluorescein; D002403:Cathepsins; D010447:Peptide Hydrolases; D003546:Cysteine Endopeptidases; D002401:Cathepsin B; C534281:CTSL protein, human; D056668:Cathepsin L; D002402:Cathepsin D; C031375:pepstatin