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Purification of Ubiquitinated p53 Proteins from Mammalian Cells
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Purification of Ubiquitinated p53 Proteins from Mammalian Cells

Published on: March 21, 2022

The C terminus of p53 binds the N-terminal domain of MDM2

Masha V Poyurovsky1, Chen Katz, Oleg Laptenko

  • 1Department of Biological Sciences, Columbia University, New York, New York, USA.

Nature Structural & Molecular Biology
|July 20, 2010
PubMed

Insights

The p53 C-terminus regulates its interaction with Mdm2, a key negative regulator. Modifications to the p53 C-terminus, like acetylation, reduce Mdm2 binding, impacting tumor suppressor activity.

Area of Science:

  • Molecular Biology
  • Cancer Research
  • Protein Interactions

Background:

  • The p53 tumor suppressor protein is regulated by its interaction with Mdm2.
  • Mdm2 acts as a negative regulator of p53, targeting it for degradation.
  • The p53 C-terminus contains sites for Mdm2-mediated ubiquitination and other post-translational modifications.

Purpose of the Study:

  • To investigate the role of the p53 C-terminus in regulating the Mdm2-p53 interaction.
  • To determine how post-translational modifications of the p53 C-terminus affect Mdm2 binding.
  • To elucidate the mechanism by which Mdm2 influences p53 binding to DNA.

Main Methods:

  • Site-directed mutagenesis to delete or mutate the p53 C-terminus.
  • In vitro binding assays using peptides and full-length proteins.
  • Cell-based assays to assess Mdm2-p53 association and DNA binding.
  • Analysis of p53 acetylation and other modifications.

Main Results:

  • Deletion, mutation, or acetylation of the p53 C-terminus decreased the Mdm2-p53 interaction.
  • Mdm2 binding to DNA was inhibited by full-length p53 but not C-terminally deleted p53.
  • A p53 C-terminal peptide directly bound to the Mdm2 N-terminus.
  • p300-acetylated p53 showed inefficient binding to Mdm2 in vitro.
  • Nutlin-3 treatment induced p53 C-terminal modifications, explaining its limited efficacy in dissociating p53-Mdm2 complexes.

Conclusions:

  • The p53 C-terminus is a critical regulatory region for the Mdm2-p53 interaction.
  • Post-translational modifications of the p53 C-terminus modulate Mdm2 binding and p53 activity.
  • Understanding these interactions is crucial for developing targeted cancer therapies.

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