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A substrate for deubiquitinating enzymes based on time-resolved fluorescence resonance energy transfer between
Robert A Horton1, Elizabeth A Strachan, Kurt W Vogel
1Invitrogen Discovery Sciences, 501 Charmany Drive, Madison, WI 53719, USA.
Abstract:
Deubiquitinating enzymes (DUBs) proteolytically cleave ubiquitin from ubiquitinated proteins, and inhibition of DUBs that rescue oncogenic proteins from proteasomal degradation is of emerging therapeutic interest. Recently, USP2 and UCH37 have been shown to deubiquitinate tumor-growth-promoting proteins, and other DUBs have been shown to be overexpressed in cancer cells. Therefore inhibition of DUBs is of interest as a potential therapeutic strategy for treating cancer. DUBs require the presence of properly folded ubiquitin protein in the substrate for efficient proteolysis, which precludes the use of synthetic peptide substrates in DUB activity assays. Because of the requirement for full-length ubiquitin, substrates suitable for use in fluorescent assays to identify or study DUB inhibitors have been difficult to prepare. We describe the development of a time-resolved fluorescence resonance energy transfer (FRET)-based DUB substrate that incorporates full-length ubiquitin that is site-specifically labeled using genetically encoded yellow fluorescent protein (YFP) and a chemically attached terbium donor. The intact substrate shows a high degree of FRET between terbium and YFP, whereas DUB-dependent cleavage leads to a decrease in FRET.
Insights
Researchers developed a novel fluorescent substrate to study deubiquitinating enzymes (DUBs). This tool enables better identification of potential cancer therapeutics by monitoring DUB activity on full-length ubiquitin substrates.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Deubiquitinating enzymes (DUBs) remove ubiquitin from proteins, playing a role in cancer by preventing proteasomal degradation of oncogenic proteins.
- Inhibiting DUBs is a promising cancer therapeutic strategy, but requires effective assays.
- Current DUB assays are limited by the need for full-length ubiquitin, hindering the development of fluorescent substrates.
Purpose of the Study:
- To develop a novel, time-resolved fluorescence resonance energy transfer (FRET)-based substrate for studying DUB activity.
- To create a tool for identifying and characterizing DUB inhibitors for cancer therapy.
Main Methods:
- Genetically encoded yellow fluorescent protein (YFP) was used for site-specific labeling of full-length ubiquitin.
- A terbium donor was chemically attached to the labeled ubiquitin to create a FRET pair.
- The developed substrate was used in time-resolved FRET assays to monitor DUB-dependent cleavage.
Main Results:
- The intact FRET substrate exhibited high FRET efficiency between terbium and YFP.
- DUB-mediated cleavage of the substrate resulted in a measurable decrease in FRET.
- This demonstrates the utility of the substrate for detecting DUB activity.
Conclusions:
- A novel, functional FRET-based substrate incorporating full-length ubiquitin has been successfully developed.
- This substrate facilitates the study of DUBs and the discovery of DUB inhibitors.
- The developed assay provides a valuable tool for advancing cancer therapeutics targeting DUBs.
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