A noninvasive cell-based assay for monitoring proteolytic activity within a specific subcellular compartment.
A 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-CHN(2). 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.
Biotechnology Research Institute, National Research Council of Canada, Montreal, Quebec H4P 2R2, Canada.
Abbes
A
Belkhiri
Viktoria
V
Lytvyn
Claire
C
Guilbault
Lucie
L
Bourget
Bernard
B
Massie
Dorit K
DK
Nägler
Robert
R
Ménard
D000595:Amino Acid Sequence; D001681:Biological Assay / Q000379:methods*; D056668:Cathepsin L; D002403:Cathepsins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D003546:Cysteine Endopeptidases; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007930:Leucine / Q000031:analogs & derivatives* / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism*; D008247:Lysosomes / Q000378:metabolism*; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D002403:Cathepsins; D003546:Cysteine Endopeptidases; C534281:CTSL protein, human; D056668:Cathepsin L; D007930:Leucine; C024974:E 64