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Updated: Jun 18, 2026
Identification of Kinase-substrate Pairs Using High Throughput Screening
Published on: August 29, 2015
Substrate screening identifies a novel target sequence for the proteasomal activity regulated by ionizing radiation
Angela Broggini-Tenzer1, Andreas Hollenstein, Zbigniew Pianowski
1Laboratory for Molecular Radiobiology, University Hospital Zurich, CH-8091 Zürich, Switzerland.
Abstract:
The screening for treatment-induced enzyme activities offers the opportunity to discover important regulatory mechanisms and the identification of potential targets for anti-cancer therapies. A novel screening technique was applied to screen substrate peptide sequences for proteolytic activities up- or down-regulated by ionizing radiation in tumor cells. One specific substrate sequence was cleaved in control cell extracts but to a smaller extent in irradiated cell extracts and investigated in detail. Based on protease-class-specific inhibitory studies and cleavage site analysis a potent warhead-inhibitor was synthesized and used to identify the proteasome as the protease of interest. The investigated sequence shows high homology to a regulatory site of nucleoporin 50, an element of the nuclear pore complex, and site specific cleavage of nucleoporin 50 was determined in vitro suggesting a novel link between the ionizing radiation-regulated proteasome and nuclear protein shuttling.
Insights
Ionizing radiation alters enzyme activity in tumor cells, revealing the proteasome
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- Enzyme activity screening can uncover regulatory mechanisms and anti-cancer drug targets.
- Ionizing radiation (IR) influences cellular processes, including enzyme regulation in tumor cells.
Purpose of the Study:
- To develop and apply a novel screening technique to identify IR-regulated proteolytic activities in tumor cells.
- To characterize a specific IR-down-regulated protease and its substrate.
Main Methods:
- A novel screening technique was used to identify substrate peptide sequences with altered proteolytic activity upon IR exposure.
- Protease-class-specific inhibitors and cleavage site analysis were employed to identify the enzyme responsible.
- A warhead-inhibitor was synthesized to pinpoint the protease.
- In vitro assays were performed to confirm substrate cleavage.
Main Results:
- A specific substrate peptide showed reduced cleavage in IR-treated cell extracts compared to controls.
- The proteasome was identified as the key protease responsible for cleaving the substrate.
- The substrate sequence shares homology with a regulatory site on nucleoporin 50 (NUP50).
- Site-specific cleavage of NUP50 by the proteasome was confirmed in vitro.
Conclusions:
- Ionizing radiation modulates proteasome activity, affecting the cleavage of specific substrates.
- The proteasome's interaction with nucleoporin 50 suggests a new regulatory pathway linking IR, proteasome function, and nuclear transport.
