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Purification of Ubiquitinated p53 Proteins from Mammalian Cells
10:55

Purification of Ubiquitinated p53 Proteins from Mammalian Cells

Published on: March 21, 2022

Topors functions as an E3 ubiquitin ligase with specific E2 enzymes and ubiquitinates p53

Rajeev Rajendra1, Diptee Malegaonkar, Pooja Pungaliya

  • 1Department of Pharmacology, The Cancer Institute of New Jersey, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, New Brunswick, NJ 08901, USA.

The Journal of Biological Chemistry
|July 13, 2004
PubMed

Insights

Human topoisomerase I- and p53-binding protein topors acts as a RING-dependent E3 ubiquitin ligase. It targets the p53 protein for degradation, similar to MDM2, suggesting a role in regulating transcription factors.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Background:

  • Topoisomerase I- and p53-binding protein (Topors) possesses a conserved N-terminal RING domain, homologous to E3 ubiquitin ligases.
  • E3 ubiquitin ligases play crucial roles in protein degradation pathways.

Purpose of the Study:

  • To investigate the E3 ubiquitin ligase activity of human Topors.
  • To identify substrates and functional consequences of Topors-mediated ubiquitination.

Main Methods:

  • In vitro ubiquitination assays using various E2 enzymes.
  • Site-directed mutagenesis to identify key residues for activity.
  • Cellular overexpression studies in human osteosarcoma cells.
  • Western blot analysis to assess p53 protein levels.

Main Results:

  • Topors functions as a RING-dependent E3 ubiquitin ligase with specific E2 enzymes (UbcH5a, UbcH5c, UbcH6).
  • A conserved tryptophan in the Topors RING domain is essential for ubiquitination.
  • Topors ubiquitinates p53 in vitro and in cellular assays.
  • Overexpression of Topors leads to proteasome-dependent degradation of p53, reducing its protein levels.

Conclusions:

  • Human Topors functions as a ubiquitin ligase, targeting p53 for degradation.
  • Topors activity is dependent on its RING domain and a specific tryptophan residue.
  • Topors may regulate multiple transcription factors, similar to its Drosophila orthologue.

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