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A Targeted Protease Substrate for a Quantitative Determination of Protease Activities in the Endolysosomal Pathway
Author(s) -
Fischer Rainer,
Bächle Daniel,
FotinMleczek Mariola,
Jung Günther,
Kalbacher Hubert,
Brock Roland
Publication year - 2006
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.200600209
Subject(s) - proteases , protease , chemistry , förster resonance energy transfer , biochemistry , proteasome , peptide , cathepsin , substrate (aquarium) , biophysics , cytoplasm , microbiology and biotechnology , proteolysis , enzyme , biology , fluorescence , ecology , physics , quantum mechanics
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.