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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

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Preferential digestion of PCNA-ubiquitin and p53-ubiquitin linkages by USP7 to remove polyubiquitin chains from

Yuji Masuda1,2, Rie Kanao3,2, Hidehiko Kawai4

  • 1From the Department of Genome Dynamics, Research Institute of Environmental Medicine, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan, masuda@riem.nagoya-u.ac.jp.

The Journal of Biological Chemistry
|January 17, 2019
PubMed

Insights

Ubiquitin-specific protease 7 (USP7) removes polyubiquitin chains from proteins like PCNA by cleaving the protein-ubiquitin bond. This mechanism, driven by USP7

Area of Science:

  • Molecular Biology
  • Biochemistry
  • Cellular Biology

Background:

  • Ubiquitin-specific protease 7 (USP7) is a key regulator of cellular pathways via its deubiquitination activity.
  • The precise molecular mechanisms by which USP7 removes ubiquitin chains from substrates are not fully understood.
  • Understanding USP7's deubiquitination mechanism is crucial for elucidating its role in various cellular processes.

Purpose of the Study:

  • To investigate the deubiquitination mechanism of Lys63-linked polyubiquitinated proliferating cell nuclear antigen (PCNA) by USP7.
  • To elucidate the substrate-specific cleavage preferences of USP7.
  • To propose a general mechanism for polyubiquitin signal removal by USP7.

Main Methods:

  • Biochemical analyses were employed to study the interaction and activity of USP7 on polyubiquitinated substrates.
  • Deubiquitination assays were performed using Lys63-linked polyubiquitinated PCNA and Lys48-linked polyubiquitinated p53.
  • Enzymatic activity and cleavage efficiency of USP7 towards different ubiquitin linkages and substrate linkages were assessed.

Main Results:

  • USP7 efficiently removes polyubiquitin chains from polyubiquitinated PCNA by preferentially cleaving the PCNA-ubiquitin linkage.
  • USP7 exhibits poor activity towards Lys63-linked ubiquitin chains, contributing to its substrate-specific cleavage.
  • Preferential cleavage of substrate-ubiquitin linkages was also observed for Lys48-linked polyubiquitinated p53 due to inefficient cleavage of Lys48-linked chains.

Conclusions:

  • USP7 employs a mechanism of preferential cleavage of substrate-ubiquitin linkages for deubiquitination.
  • The enzyme's activity is influenced by the type of ubiquitin linkage and the specific substrate.
  • This study provides insights into how USP7 removes polyubiquitin signals, impacting cellular pathway regulation.
Keywords:
DNA damage responsedeubiquitylation (deubiquitination)enzyme kineticsp53proliferating cell nuclear antigen (PCNA)

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