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Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

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Homogeneous, bioluminescent proteasome assays.

Martha A O'Brien¹, Richard A Moravec, Terry L Riss

¹Promega Corporation, Madison, WI, USA.

Methods in Molecular Biology (Clifton, N.J.)

|January 8, 2008

Summary

Researchers developed a novel bioluminescent assay to monitor proteasome activity, overcoming limitations of fluorescent methods for cancer drug discovery. This sensitive assay is ideal for screening proteasome inhibitors.

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Area of Science:

Biochemistry
Molecular Biology
Drug Discovery

Background:

The ubiquitin-proteasome pathway is crucial for protein degradation and cellular homeostasis.
The proteasome is a validated therapeutic target for cancer treatment.
Existing fluorogenic assays for proteasome activity have limitations, including high background and interference from cellular autofluorescence.

Purpose of the Study:

To develop a sensitive, homogeneous, and robust assay for monitoring proteasome activity.
To overcome the limitations of current fluorogenic assays for proteasome inhibitor screening.
To establish bioluminescent assays for all three major proteasome activities.

Main Methods:

Development of a homogeneous, bioluminescent assay using peptide-conjugated aminoluciferin substrates and stabilized luciferase.
Assays were developed for chymotrypsin-like, trypsin-like, and caspase-like proteasome activities using purified proteasome.
Application of the technology to a cellular assay for chymotrypsin-like activity with a membrane permeabilization step.
Assays were validated in 96- and 384-well plate formats.

Main Results:

Successful development of homogeneous bioluminescent assays for all three proteasome activities.
Demonstrated utility of the bioluminescent assay in a cellular context for chymotrypsin-like activity.
The "add and read" format provides a simple and rapid method for proteasome activity monitoring.

Conclusions:

The novel bioluminescent assay offers a sensitive and rapid alternative to fluorogenic substrates for proteasome activity monitoring.
This assay format is well-suited for high-throughput screening of proteasome inhibitors in cancer research.
The technology addresses key limitations of existing assays, facilitating drug discovery efforts targeting the proteasome.