Profiling proteasome activity in tissue with fluorescent probes
Celia R Berkers1, Fijs W B van Leeuwen, Tom A Groothuis
1Division of Cellular Biochemistry, Tumor Biology, and Immunology, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
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.
Insights
New fluorescent probes enable sensitive profiling of proteasome activity in living cells. These tools aid in evaluating proteasome inhibitor efficacy for cancer treatment and understanding organ-specific proteasome regulation.
Area of Science:
- Biochemistry
- Cell Biology
- Pharmacology
Background:
- Proteasome inhibitors are crucial in treating multiple myeloma and other cancers.
- Accurate methods are needed to assess proteasome inhibitor specificity and efficacy in patients.
Purpose of the Study:
- To develop and validate sensitive fluorescent reporters for profiling proteasome activity in living cells.
- To compare the specificity and utility of different fluorescent probes for proteasome activity measurement.
Main Methods:
- Synthesis and biochemical validation of novel fluorescent proteasome activity reporters.
- Application of probes in gel-based assays, confocal microscopy, and flow cytometry.
- Profiling proteasome activity in various mouse tissues, controlling for postlysis artifacts.
Main Results:
- Seven synthesized probes successfully labeled proteasomes in cell lysates, with specificity influenced by the fluorescent dye.
- Two differentially labeled probes demonstrated suitability for live-cell proteasome activity analysis.
- Proteasome subunit activity was found to be regulated in an organ-specific manner in mouse tissues.
Conclusions:
- Developed fluorescent reporters offer a sensitive and versatile tool for studying proteasome activity in living cells.
- These methods can be applied to assess proteasome inhibitor pharmacodynamics in vivo.
- Organ-specific regulation of proteasome activity was elucidated using these novel reporters.
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