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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.

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Figure 1: Fusion to ubiquitin destabilizes firefly luciferase through proteasome degradation.
Figure 2: Inhibition of the proteasome produces concentration-dependent increases in bioluminescence from Ub-FL.
Figure 3: Bioluminescence from Ub-FL shows time-dependent increases in response to inhibition of the proteasome.
Figure 4: In vivo bioluminescence imaging of Ub-FL monitors proteasome function and inhibition in living mice.
Figure 5: Imaging temporal inhibition of the proteasome with bortezomib in vivo.

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Acknowledgements

This work was funded by National Institutes of Health grant P50 CA94056.

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Correspondence to David Piwnica-Worms.

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Luker, G., Pica, C., Song, J. et al. Imaging 26S proteasome activity and inhibition in living mice. Nat Med 9, 969–973 (2003). https://doi.org/10.1038/nm894

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