Anne Swanson
Lab Experience: Working with p53
The protein p53 is an important tumor-suppressor protein that is often referred to as “the guardian of the genome.” When activated by stressors, such as DNA damage, p53 responds by activating genes that induce cell-cycle arrest, apoptosis, and DNA repair. However, when p53 is mutated, cancerous phenotypes often result. Studying p53 is important since p53 mutations occur in half of all cancers (Seton-Rogers 2006).
Last semester I participated in a research project that investigated the effects of several p53 mutants. The study was designed as an introductory research experience for undergraduates, modeled after a Stanford University study of p53 mutants by Hekmat-Scafe et al. (2017). When p53 is mutated, it can lose its ability to bind to these important target genes. We produced strains of yeast with response elements normally bound by p53. We then incorporated these yeast samples with mutant-p53 DNA. We performed filter-lift assays on each yeast sample, as well as controls, to determine how well each mutant transactivates its target proteins. In addition, we performed a Western blot to assess the stability of each mutant-p53 protein.
Bykov V. J., Wiman K. G. (2014) Mutant p53 reactivation by small molecules makes its way to the clinic. FEBS Letters, 588, 2622–7.
Hekmat‐Scafe, D. S., Brownell, S. E., Seawell, P. C., Malladi, S., Imam, J. F. C., Singla, V., ... & Stearns, T. 2017. Using yeast to determine the functional consequences of mutations in the human p53 tumor suppressor gene: An introductory course‐based undergraduate research experience in molecular and cell biology. Biochemistry and Molecular Biology Education, 45(2), 161-178.
Seton-Rogers, S. (2006) Putting p53 in context. Nature Reviews Cancer, 6, 423.
All five mutations resulted in transactivation defects, but the severity of each defect varied. The Western blot indicated decreased p53 protein stability for three of the mutants (no data was produced for the other two). These results have implications regarding mutant location and drug therapies. In order to most effectively develop cancer treatments, it is necessary to understand and target the specific genetic mechanisms that contribute to cancer development. For example, studies investigating the structural and functional changes that result from the Y220C mutation, one of the five mutations researched in this study, have implicated it is a promising target for drug discovery. The surface crevice that the mutation creates can be targeted by small molecules in order to restabilize the protein and regain function (Bykov 2014).
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