A noninvasive cell-based assay for monitoring proteolytic activity within a specific subcellular compartment

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DOIResolve DOI: http://doi.org/10.1006/abio.2002.5706
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TypeArticle
Journal titleAnalytical Biochemistry
ISSN0003-2697
Volume306
Issue2
Pages237246; # of pages: 10
Subjectpha; amino acid sequence; biological assay; cathepsins; cysteine endopeptidases; green fluorescent proteins; hela cells; humans; hydrogen-ion concentration; leucine; luminescent proteins; lysosomes; molecular sequence data; recombinant fusion proteins; spectrometry; fluorescence
AbstractA noninvasive cell-based assay has been developed to monitor the proteolytic activity of cathepsin L within a specific subcellular compartment, the lysosome. The green fluorescent protein (GFP) of Aequorea victoria was selected as a substrate. Targeting to lysosomes was achieved by fusing GFP to preprocathepsin L, which also ensures colocalization of the enzyme and the substrate. Stably transfected HeLa-rtTA (reverse tetracycline-controlled transactivator) cells were induced with doxycycline and cultured in the presence of various concentrations of cysteine protease inhibitors for 48 h. In the absence of inhibitor, proteolytic degradation of GFP leads to loss of fluorescence, which is due almost exclusively to the action of recombinant cathepsin L. However, a dose-dependent increase of GFP fluorescence is observed for cells treated with the potent cathepsin L inhibitor benzyloxycarbonyl-LeuLeuTyr-CHN₂. Fluorescence is also observed when GFP is fused to an inactive preprocathepsin L (C25A mutant). Targeting of GFP to an acidic cellular compartment can destabilize the protein and render it susceptible to proteolytic degradation. The approach should be generally applicable for proteases localized in acidic environments. Such an assay can be of great value in validating the participation of a specific enzyme in a given process or in testing the ability of putative inhibitors to reach their intracellular target.
Publication date
LanguageEnglish
AffiliationNRC Biotechnology Research Institute; National Research Council Canada
Peer reviewedNo
Identifier10269630
NRC number44835
NPARC number3539232
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Record identifierf457e2d4-1694-4323-aa7d-4b64d98bdd6b
Record created2009-03-01
Record modified2016-05-09
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