Imaging 26S proteasome activity and inhibition in living mice

Abstract

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.

Factors affecting human T cell engraftment, trafficking, and associated xenogeneic graft-vs-host disease in NOD/SCID β2mnull mice

“…[46] Bioluminescence Imaging of Animals Human T cell trafficking in NOD/SCID-β2m null recipients was assessed non-invasively by bioluminescence with an IVIS 100 CCD camera (Xenogen, Alameda, CA, USA) as described. [47,48] Briefly, mice were injected i.p. with D-luciferin (150 μg/g in PBS) and imaged 10 min later with the IVIS (1-60 sec exposure, binning 8, f-stop 1, FOV 15 cm).…”

Imaging reversal of multidrug resistance in living mice with bioluminescence: MDR1 P-glycoprotein transports coelenterazine

“…The gene has been cloned and sequenced (7) and used extensively in reporter gene assays in bacteria, yeast, plant, and mammalian cells (8) for bioluminescence resonance energy transfer studies and for high throughput drug screening (9-11). Rluc provides a kinetically rapid, alternative bioluminescence signal to firefly (Photinus pyralis) luciferase and, when coexpressed, enables dual reporter readouts.A rapidly evolving area of biomedical research, use of bioluminescence as an optical marker for gene expression has been recently extended to noninvasive, real-time analysis of molecular events in intact cells and living animals (12)(13)(14). Although previous data suggested that Rluc could be used for reporter imaging in mice in vivo (15), limited utility and poor bioavailability of coelenterazine was reported in a mouse model of viral infection with an Rluc reporter strain of herpes simplex virus 1 (16).…”

“…A rapidly evolving area of biomedical research, use of bioluminescence as an optical marker for gene expression has been recently extended to noninvasive, real-time analysis of molecular events in intact cells and living animals (12)(13)(14). Although previous data suggested that Rluc could be used for reporter imaging in mice in vivo (15), limited utility and poor bioavailability of coelenterazine was reported in a mouse model of viral infection with an Rluc reporter strain of herpes simplex virus 1 (16).…”

Proteasome properties of hemocytes differ between the whiteleg shrimp Penaeus vannamei and the brown shrimp Crangon crangon (Crustacea, Decapoda)

“…Epoxomicin (PeptaNova, 4381), gliotoxin (Applichem, APPA7665), lactacystin (Cayman chemical company, Cas 133343-34-7), and MG132 (Sigma, C2211) were dissolved in DMSO, aliquoted, and stored at −20°C. The final concentrations of inhibitors in the assays were epoxomicin 50 mmol L −1 (Götze and Saborowski 2011), lactacystin 1 mmol L −1 , gliotoxin 100 mmol L −1 (Kroll et al 1999), and MG132 25 mmol L −1 (Luker et al 2003). Enzymatic assays were performed as described above (n = 3).…”