Document Detail


A targeted protease substrate for a quantitative determination of protease activities in the endolysosomal pathway.
MedLine Citation:
PMID:  16871600     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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:
Rainer Fischer; Daniel Bächle; Mariola Fotin-Mleczek; Günther Jung; Hubert Kalbacher; Roland Brock
Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Chembiochem : a European journal of chemical biology     Volume:  7     ISSN:  1439-4227     ISO Abbreviation:  Chembiochem     Publication Date:  2006 Sep 
Date Detail:
Created Date:  2006-09-04     Completed Date:  2006-10-30     Revised Date:  2009-11-19    
Medline Journal Info:
Nlm Unique ID:  100937360     Medline TA:  Chembiochem     Country:  Germany    
Other Details:
Languages:  eng     Pagination:  1428-34     Citation Subset:  IM    
Affiliation:
Eberhard Karls University Tübingen, Department of Molecular Biology, Interfaculty Institute for Cell Biology, Auf der Morgenstelle 15, 72076 Tübingen, Germany.
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MeSH Terms
Descriptor/Qualifier:
Catalysis / 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:
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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