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Updated: Aug 25, 2026
Purification of Ubiquitinated p53 Proteins from Mammalian Cells
Published on: March 21, 2022
A post-ubiquitination role for MDM2 and hHR23A in the p53 degradation pathway
Chrystelle Brignone1, Kathleen E Bradley, Alexei F Kisselev
1Department of Cancer Biology, University of Massachusetts Medical School, Worcester, MA 01605, USA.
Abstract:
Abrogation of ubiquitin/proteasome-dependent turnover of p53 is critical for its activation. UbL-UBA proteins, including human homolog of Rad23 (hHR23) proteins, may regulate proteasomal degradation of substrates such as p53, due to their ability to interact with both ubiquitinated substrates and the proteasome. siRNA-mediated depletion of hHR23A or hHR23B in human cell lines accelerated p53 degradation. In contrast, overexpression of hHR23 proteins led to the accumulation of ubiquitinated p53, and purified hHR23 proteins also blocked p53 proteasome degradation in vitro. An hHR23-MDM2 complex was identified, suggesting that MDM2 and hHR23 cooperate in the regulation of p53 proteasome degradation. Consistent with this hypothesis, an MDM2 mutant that demonstrated increased binding in vivo to hHR23A was able to ubiquitinate, but not degrade p53. Moreover, the defective phenotype of this MDM2 mutant was rescued by siRNA knockdown of hHR23A. Our data indicate that MDM2 acts at a step in the p53 degradation pathway after ubiquitination, to counteract hHR23 inhibition of p53 turnover. Moreover, our data suggest the possibility that ubiquitin ligase/UbL-UBA protein complexes, as exemplified by the MDM2/hHR23 complex, may serve a general role in regulating substrate degradation by the proteasome.
Insights
Human homolog of Rad23 (hHR23) proteins regulate p53 proteasome degradation. MDM2 and hHR23 cooperate to control p53 turnover, revealing a general mechanism for proteasomal substrate regulation.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- The turnover of the p53 protein, a critical tumor suppressor, is tightly regulated by the ubiquitin/proteasome system.
- Ubiquitin-like (UbL) and ubiquitin-associated (UBA) domain proteins, such as human homolog of Rad23 (hHR23), interact with ubiquitinated substrates and the proteasome, suggesting a role in substrate degradation.
Purpose of the Study:
- To investigate the role of hHR23 proteins in the proteasomal degradation of p53.
- To elucidate the interplay between hHR23 proteins, MDM2, and p53 regulation.
Main Methods:
- RNA interference (siRNA) to deplete hHR23A or hHR23B in human cell lines.
- Overexpression of hHR23 proteins.
- In vitro degradation assays using purified hHR23 proteins.
- Identification of hHR23-MDM2 complexes.
- Analysis of an MDM2 mutant with altered hHR23 binding.
Main Results:
- Depletion of hHR23A or hHR23B accelerated p53 degradation, while overexpression led to the accumulation of ubiquitinated p53.
- Purified hHR23 proteins inhibited p53 proteasome degradation in vitro.
- An hHR23-MDM2 complex was identified, indicating cooperation in p53 regulation.
- An MDM2 mutant binding hHR23A ubiquitinated p53 but failed to degrade it, a phenotype rescued by hHR23A knockdown.
Conclusions:
- MDM2 functions downstream of ubiquitination in the p53 degradation pathway, counteracting hHR23's inhibition of p53 turnover.
- Ubiquitin ligase/UbL-UBA protein complexes, like MDM2/hHR23, may play a general role in regulating proteasomal substrate degradation.
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