| A targeted protease substrate for a quantitative determination of protease activities in the endolysosomal pathway. | |
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MedLine Citation:
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PMID: 16871600 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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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. |
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Authors:
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Rainer Fischer; Daniel Bächle; Mariola Fotin-Mleczek; Günther Jung; Hubert Kalbacher; Roland Brock |
Publication Detail:
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Type: Journal Article; Research Support, Non-U.S. Gov't |
Journal Detail:
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Title: Chembiochem : a European journal of chemical biology Volume: 7 ISSN: 1439-4227 ISO Abbreviation: Chembiochem Publication Date: 2006 Sep |
Date Detail:
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Created Date: 2006-09-04 Completed Date: 2006-10-30 Revised Date: 2009-11-19 |
Medline Journal Info:
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Nlm Unique ID: 100937360 Medline TA: Chembiochem Country: Germany |
Other Details:
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Languages: eng Pagination: 1428-34 Citation Subset: IM |
Affiliation:
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Eberhard Karls University Tübingen, Department of Molecular Biology, Interfaculty Institute for Cell Biology, Auf der Morgenstelle 15, 72076 Tübingen, Germany. |
Export Citation:
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| MeSH Terms | |
Descriptor/Qualifier:
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Catalysis
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drug effects Cathepsin B / metabolism Cathepsin D / metabolism Cathepsin L Cathepsins / metabolism Cysteine Endopeptidases / metabolism Endocytosis / drug effects Endosomes / enzymology* Enzyme Inhibitors / pharmacology Fluoresceins / chemistry Fluorescence Resonance Energy Transfer Fluorescent Dyes / chemistry Hela Cells Humans Lysosomes / enzymology* Oligopeptides / chemistry Pepstatins / pharmacology Peptide Hydrolases / metabolism* Peptides / chemical synthesis, metabolism* Protease Inhibitors / pharmacology Rhodamines / chemistry Spectrometry, Fluorescence Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization Subcellular Fractions / chemistry, metabolism Substrate Specificity |
| Chemical | |
Reg. No./Substance:
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0/5-carboxytetramethylrhodamine succinimidyl ester; 0/Enzyme Inhibitors; 0/Fluoresceins; 0/Fluorescent Dyes; 0/Oligopeptides; 0/Pepstatins; 0/Peptides; 0/Protease Inhibitors; 0/Rhodamines; 0/nonaarginine; 3301-79-9/6-carboxyfluorescein; 39324-30-6/pepstatin; EC 3.4.-/Cathepsins; EC 3.4.-/Peptide Hydrolases; EC 3.4.22.-/Cysteine Endopeptidases; EC 3.4.22.1/Cathepsin B; EC 3.4.22.15/CTSL1 protein, human; EC 3.4.22.15/Cathepsin L; EC 3.4.23.5/Cathepsin D |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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