p53 as a molecular time bomb
: a biochemical approach to dissect its regulation by posttranslational modifications

Student thesis: Doctoral ThesisDoctor of Philosophy

Abstract

p53, one of the most important tumour suppressor proteins, keeps aberrant cells in check. Its inactivation is a prerequisite for the growth of any tumour. The mechanisms of how different cellular stresses activate p53 and how p53 integrates stress signals to induce either cell cycle arrest or cell death remain elusive despite decades of extensive research. An understanding of how the p53 bomb is kindled to induce the removal of damaged cells may enable scientists to address and utilise its function in a more targeted way for therapeutic purposes in the future. In this work, this complex system was tackled using a reductionist approach by establishing an in vitro system that allowed for the biochemical characterisation of p53 activation. At the core of this system was a refolding process that provided access to the active tetrameric species of p53 and that was compatible with recombinantly expressed, posttranslational modification (PTM)-free p53 molecules as well as semi-synthetic p53 variants that contained site-specific PTMs. It was demonstrated on the basis of these homogeneous p53 tetramers that damage-associated Ser20 phosphorylated p53 (Ser20ph) directly enhanced p300-mediated acetylation of p53 in the distal C-terminus 2- to 3-fold; this result highlighted an interplay of PTMs in different domains of p53 and the power of this approach for mechanistic insights into p53 activation cascades. The complexity of p53 function becomes especially apparent in its switch in cellular response upon prolonged stress duration. How is such a switch mediated? Spontaneous asparagine (Asn) deamidation was suggested here as the solution to this conundrum. The half-life for Asn29 deamidation was determined to be ∼10 days under physiologically relevant conditions. This timer would allow cells a first repair attempt, but could change the stress response upon prolonged times of p53 activation on biologically relevant time scales. The location of this Asn residue within the N-terminal protein-protein interaction hotspot of p53 hinted at a regulatory role for this PTM by controlling differential binding partner recruitment. It was shown that Asn deamidation directly resulted in a reduced interaction with the p53 negative regulator MDM2 on the peptide level, suggesting a phosphorylation-independent mode to prevent p53 degradation. Furthermore, enhanced acetylation of isoAspartate (isoAsp)-bearing p53 tetramers by p300 was observed, providing a path to initiate a change in the p53-mediated cellular response. Overall, these studies offer a new approach to unravel p53 regulation as a function of its PTM state, which is based on quantitative, direct measurements, utilising the active, fulllength species. Many further extensions to the herein described toolset can be imagined for the future.
Date of Award1 Mar 2021
Original languageEnglish
Awarding Institution
  • King's College London
SupervisorManuel Muller (Supervisor) & Ulrike Eggert (Supervisor)

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