Imaging 26S proteasome activity and inhibition in living mice.
The ubiquitin-proteasome pathway is the central mediator of regulated proteolysis in cells, and defects in this pathway are associated with cancer and neurodegenerative diseases. To assess 26S proteasome function in living animals, we developed a ubiquitin-luciferase reporter for bioluminescence imaging. The reporter was degraded rapidly under steady-state conditions and stabilized in a dose- and time-dependent manner in response to proteasome inhibitors. Using bioluminescence imaging after one dose of the chemo-therapeutic proteasome inhibitor bortezomib (PS-341), proteasome function in tumor xenografts was blocked within 30 min and returned to nearly baseline by 46 h. After a 2-week regimen of bortezomib, however, imaging of target tumors showed significantly enhanced proteasome inhibition that no longer returned to baseline. The ubiquitin-luciferase reporter enables repetitive tissue-specific analysis of 26S proteasome activity in vivo and should facilitate development and validation of proteasome inhibitors in mouse models, as well as investigations of the ubiquitin-proteasome pathway in disease pathogenesis.
Luker
Gary D
GD
Molecular Imaging Center, Mallinckrodt Institute of Radiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Pica
Christina M
CM
Song
Jiling
J
Luker
Kathryn E
KE
Piwnica-Worms
David
D
D000111:Acetylcysteine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D001897:Boronic Acids / Q000494:pharmacology; D000069286:Bortezomib; D001517:Coleoptera; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D007976:Leupeptins / Q000494:pharmacology; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008163:Luminescent Measurements; D008297:Male; D051379:Mice; D008819:Mice, Nude; D008967:Molecular Biology / Q000379:methods*; D010447:Peptide Hydrolases / Q000032:analysis* / Q000187:drug effects / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D011719:Pyrazines / Q000494:pharmacology; D014162:Transfection; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism; D023041:Xenograft Model Antitumor Assays
D001897:Boronic Acids; D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D011719:Pyrazines; D025801:Ubiquitin; C067713:lactacystin; D000069286:Bortezomib; D008156:Luciferases; D010447:Peptide Hydrolases; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde; D000111:Acetylcysteine