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Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer
Published on: May 26, 2014
22.7K
Homogeneous, bioluminescent proteasome assays.
Martha A O'Brien1, Richard A Moravec, Terry L Riss
1Promega Corporation, 2800 Woods Hollow Road, Madison, WI, 53711, USA, martha.obrien@promega.com.
Methods in Molecular Biology (Clifton, N.J.)
|October 14, 2014
Summary
A new bioluminescent assay enables sensitive, single-step measurement of proteasome activity in cells. This homogeneous method is ideal for high-throughput screening of proteasome inhibitors for cancer and neurodegenerative diseases.
Area of Science:
- Biochemistry
- Molecular Biology
- Drug Discovery
Background:
- The ubiquitin-proteasome pathway is crucial for protein degradation and cellular regulation.
- Proteasome dysfunction is linked to cancer, neurodegenerative, and cardiovascular diseases.
- Existing fluorogenic assays for proteasome activity have limitations, including high background and interference.
Purpose of the Study:
- To develop a homogeneous, bioluminescent assay for monitoring proteasome activity.
- To create a cell-based assay suitable for high-throughput screening (HTS).
- To enable sensitive measurement of distinct proteasome catalytic activities.
Main Methods:
- Developed a homogeneous, bioluminescent method using peptide-conjugated aminoluciferin substrates and stabilized luciferase.
- Incorporated a selective membrane permeabilization step for cell-based assays.
- Designed assays to measure chymotrypsin-like, trypsin-like, and caspase-like proteasome activities.
Main Results:
- The new method provides sensitive, single-step, cell-based assays for each proteasome catalytic activity.
- Eliminates the need for cell extract preparation, simplifying the assay workflow.
- Demonstrated adequate sensitivity for 96- and 384-well plate formats.
Conclusions:
- The developed bioluminescent assay is a robust and rapid tool for proteasome activity monitoring.
- The "add and read" format is ideal for high-throughput screening of proteasome inhibitors.
- This assay facilitates the development of therapeutics targeting proteasome-related diseases.
