Medizinische Universität Graz - Research portal
Selected Publication:
Michenthaler, H.
Establishment of a BioID system to study the p53 interactome under starvation.
[ Diplomarbeit/Master Thesis (UNI) ] TU Graz; 2021.
FullText
- Authors Med Uni Graz:
- Michenthaler Helene
- Advisor:
- Prokesch Andreas
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- Abstract:
- P53 is among the best studied molecules in cancer biology due to its tumor suppressor function. Through many years of studying its functions, several molecular mechanisms have been discovered in the last decades and over 1000 proteins have been identified that interact with p53. In recent studies, we showed that p53 stabilization is necessary for starvation-mediated sensitization to cancer therapy in resistant hepatocellular carcinoma cells. To screen for p53 interaction partners in HepG2 cells under starvation, we established a proteomics tool for protein-proximity labelling, based on the BioID system. We designed fusion proteins expressing wild-type p53, coupled to a promiscuous biotin ligase (miniTurbo) and harbouring subcellular localization signals to identify compartment-specific interaction partners in response to a starvation stimulus. In this thesis, cloning and production of BioID fusion proteins was conducted. Furthermore, the fusion proteins were validated by expressing them in p53-deficient HepG2 cells and optimal experimental conditions (i.e. biotin labelling, pulldown of labelled proteins, and localization of the fusion proteins) were derived. Finally, a mass spectrometry pilot run was performed as a proof-of-principle showing successful identification of biotinylated proteins and starvation-specific p53 interaction partners. This dataset revealed both already known p53 interaction partners as positive control as well as novel interaction partners. Hence, the work of this thesis established an invaluable system to decipher the p53 interactome under certain cellular stresses and will help to define the mechanism of p53 stabilization under nutrient deprivation.