Isolation of the Portable p53 Degron to Incorporate into an Mdm2 Reporter (2014)

Undergraduates: Lukas Dumberger, Greg Woss Adam T. Melvin

Faculty Advisor: Marcey Waters
Department: Biology

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The protein p53 plays a role in cellular functions such as DNA repair, cell cycle modulation, and apoptosis. Mutation or inactivation of p53 is a hallmark in many cancer types, affecting up to 22 million people. About half of these patients exhibit decreased p53 levels due to overexpression of its regulatory enzyme murine double minute 2 (MDM2), which decreases p53 levels by facilitating its proteasomal degradation. Due to vital involvement in cellular function, p53 and MDM2 have become targets for new therapeutic designs. As such, there is a need for reporters on MDM2 activity and p53 control via the ubiquitin proteasome system (UPS). Such reporters require the incorporation of a degradation sequence (degron) of p53, which targets the probe to its functional destination, the UPS. This project used a shotgun approach to create a fragment library of a p53 domain, necessary for MDM2 mediated degradation, to identify this minimal degron. Substrates were synthesized using Solid Phase Peptide Synthesis, purified with High Performance Liquid Chromatography (HPLC), and tested via an in vitro assay for their degree of ubiquitination to identify the smallest functional degron. Gel electrophoresis of assay samples demonstrated two substrates that were strongly ubiquitinated and are hypothesized to contain the smallest functional degron. In order to obtain more quantifiable results about substrate ubiquitination, further work is being done to analyze assay results with analytical HPLC.