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Stanford BIO 230 - Imaging 26S proteasome activity and inhibition in living mice

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TECHNICAL REPORTSThe ubiquitin-proteasome pathway is the central mediator ofregulated proteolysis in cells, and defects in this pathway areassociated with cancer and neurodegenerative diseases. Toassess 26S proteasome function in living animals, wedeveloped a ubiquitin-luciferase reporter for bioluminescenceimaging. The reporter was degraded rapidly under steady-stateconditions and stabilized in a dose- and time-dependentmanner in response to proteasome inhibitors. Usingbioluminescence imaging after one dose of the chemo-therapeutic proteasome inhibitor bortezomib (PS-341),proteasome function in tumor xenografts was blocked within30 min and returned to nearly baseline by 46 h. After a 2-weekregimen of bortezomib, however, imaging of target tumorsshowed significantly enhanced proteasome inhibition that nolonger returned to baseline. The ubiquitin-luciferase reporterenables repetitive tissue-specific analysis of 26S proteasomeactivity in vivo and should facilitate development andvalidation of proteasome inhibitors in mouse models, as well asinvestigations of the ubiquitin-proteasome pathway in diseasepathogenesis.The 26S proteasome degrades proteins that control essential signalingpathways in normal physiology. Aberrant proteasome-dependent pro-teolysis is associated with pathophysiology of malignancies, neurode-generative disorders, type I diabetes and cachexia1,2.Recently, theproteasome has emerged as a new molecular target for therapy. In ani-mal models, proteasome inhibition reduces reperfusion injury aftermyocardial ischemia3or stroke4.Blocking proteasome functionretards growth of several tumor types in mouse xenograft models andenhances efficacy of established chemotherapeutic drugs and ionizingradiation5.In a phase 2 clinical trial of the proteasome inhibitor borte-zomib (Velcade, formerly known as PS-341), most patients withrefractory, relapsed multiple myeloma showed improvement or stabi-lization of disease, and 20% achieved durable, complete remission6.The intact 26S proteasome consists of a 19S regulatory particle thatbinds ubiquitin chains, unfolds substrates and translocates proteinsinto a 20S catalytic core7,8.Ubiquitin-like proteins, ubiquitin ligases,deubiquitinating enzymes and adaptor molecules also interact withthe intact proteasome and regulate degradation of substrates, butmany of these proteins are lost during conventional purification ofproteasomes for in vitro assays9.In animal models and patients, pro-teasome activity is typically measured in blood samples with an assayof the isolated 20S core particle rather than the intact 26S protea-some10.These methods do not account for the effects of the 19S regu-latory particle or other associated proteins and may not accuratelydetermine activity of the intact 26S proteasome in living animals.Additionally, analyses of proteasome function in blood samples maynot accurately reflect proteolysis within a tumor or other target tissue.RESULTSFirefly luciferase reporter of proteasome activityTo directly assay intact 26S proteasome activity in living animals, weengineered a ubiquitin-luciferase bioluminescence imaging reporter(Ub-FL) by fusing the N terminus of firefly luciferase to four copies ofa mutant ubiquitin (ubiquitin G76V) that resists cleavage by ubiquitinhydrolases11.In transiently transfected HeLa cells, Ub-FL producedonly 5% of the bioluminescence of unfused firefly luciferase underbaseline steady-state conditions (Fig. 1a). Luciferase activity from Ub-FL increased significantly after 6 h of treatment with the proteasomeinhibitor MG132 (25 µM; P < 0.01) and became equivalent to fireflyluciferase by 8 h. MG132 did not affect the activity of firefly luciferase,showing that the tetraubiquitin fusion causes degradation of luciferasein the proteasome. Ub-FL activity during the first 4 h of treatment var-ied between experiments but was consistently low, and a delay betweenproteasomal inhibition and maximum bioluminescence was uni-formly observed in both transiently and stably transfected cells (seebelow). The time between addition of MG132 and enhanced Ub-FLactivity reflects the net kinetics of stabilization of previously translatedUb-FL and synthesis of new reporter. Bioluminescence also may beimpacted by incompletely defined effects of proteasome inhibitors onformation of transcription complexes and possibly translation ofreporter proteins such as luciferase12,13.We used clones of HeLa cells stably transfected with Ub-FL, fireflyluciferase, or vector control to further characterize degradation of Ub-FL. In cells incubated with cycloheximide to inhibit protein synthesis,bioluminescence from Ub-FL decreased by approximately 90% after15 min and was undetectable by 2 h (Fig. 1b). Luciferase activity infirefly luciferase transfectants remained at approximately 75% of con-trol values after 4 h. As expected, we did not detect luciferase activity invector control transfectants (data not shown).1Molecular Imaging Center, Mallinckrodt Institute of Radiology and 2Department of Molecular Biology and Pharmacology, Washington University School of Medicine,St. Louis, Missouri 63110, USA. Correspondence should be addressed to D.P.-W. ([email protected]).Imaging 26S proteasome activity and inhibition in living miceGary D Luker1,Christina M Pica1,Jiling Song1,Kathryn E Luker1& David Piwnica-Worms1,2NATURE MEDICINE VOLUME 9 |NUMBER 7 |JULY 2003 969NATURE MEDICINE VOLUME 9 |NUMBER 7 |JULY 2003 969© 2003 Nature Publishing Group http://www.nature.com/naturemedicineTECHNICAL REPORTSEffects of proteasome inhibitors on Ub-FL in cultured cellsTo determine whether Ub-FL could detect relative differences in 26Sproteasome activity, we treated cells stably transfected with Ub-FL andfirefly luciferase with increasing doses of compounds that reversibly(MG132 and bortezomib) or irreversibly (lactacystin) inhibit the pro-teasome14.Each compound produced concentration-dependentincreases in bioluminescence from Ub-FL (Fig. 2a–c), and peak activ-ity of Ub-FL was nearly equal to that of firefly luciferase. Half-maximaleffective concentration (EC50) values were approximately 4 µM and 1µM for MG132 and lactacystin, respectively (Fig. 2a,b). The apparentEC50value for bortezomib was 0.06 µM, although this value was not atrue EC50because bortezomib-induced Ub-FL activity did not plateauat any tested concentration (Fig. 2c). Treatment with PD150606 (400 nM), a calpain inhibitor15, did not change bioluminescence fromUb-FL (data not


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Stanford BIO 230 - Imaging 26S proteasome activity and inhibition in living mice

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