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Profiling Proteasome Activity in Tissue with Fluorescent Probes

Berkers, Celia R., van Leeuwen, Fijs W. B., Groothuis, Tom A., Peperzak, Victor, van Tilburg, Erica W., Borst, Jannie, Neefjes, Jacques J., Ovaa, Huib·2007·Molecular Pharmaceutics
DOI 10.1021/mp0700256
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Abstract

With proteasome inhibitors in use in the clinic for the treatment of multiple myeloma and with clinical trials in progress investigating the treatment of a variety of hematologic and solid malignancies, accurate methods that allow profiling of proteasome inhibitor specificity and efficacy in patients are in demand. Here, we describe the development, full biochemical validation, and comparison of fluorescent proteasome activity reporters that can be used to profile proteasome activities in living cells with high sensitivity. Seven of the synthesized probes tested label proteasomes in lysates, although the fluorescent dye used affects their specificity. Two differentially labeled probes tested are suitable for studying proteasome activity in living cells by gel-based assays, by confocal laser scanning microscopy, and by flow cytometry. We established methods using these fluorescent reporters to profile proteasome activity in different mouse tissues, carefully avoiding postlysis artifacts, and we show that proteasome subunit activity is regulated in an organ-specific manner. The techniques described here could be used to study in vivo pharmacological properties of proteasome inhibitors.

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Metadata

Authors
Berkers, C. R., van Leeuwen, F. W. B., Groothuis, T. A., Peperzak, V., van Tilburg, E. W., Borst, J., Neefjes, J. J., & Ovaa, H.
Affiliations
Divisions of Cellular Biochemistry, Tumor Biology, and Immunology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands
Venue
Molecular Pharmaceutics
Publisher
American Chemical Society (ACS)
Published
2007-08-21
Volume
4
Issue
5
Pages
739-748
ISSN
  • 1543-8384
  • 1543-8392
DOI
10.1021/mp0700256
Cited by
102
References
2
Language
en

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