C-terminal region of USP7/HAUSP is critical for deubiquitination activity and contains a second mdm2/p53 binding site

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Ubiquitin-specific protease 7 (USP7), also known as HAUSP, regulates p53 levels by deubiquitinating mdm2 and p53.
• The N-terminal TRAF-like domain of USP7 was previously thought to be crucial for mdm2/p53 binding.

Purpose of the Study:

• To investigate the functional role of USP7 domains in deubiquitination and substrate binding.
• To identify the minimal catalytic domain and additional binding sites of USP7.

Main Methods:

• Site-directed mutagenesis to delete the N-terminal TRAF-like domain of USP7.
• Enzyme kinetics assays using Ub-AFC, Ub(n)-mdm2, and Ub(n)-p53 substrates.
• Glutathione S-transferase (GST) pull-down assays to map protein-protein interaction domains.

Main Results:

• Deletion of the USP7 N-terminal TRAF-like domain did not impair deubiquitination of mdm2 and p53.
• The minimal functional USP7 enzyme spans amino acids 208-1102, retaining proteolytic activity.
• The C-terminal domain of USP7 contains specific binding sites for mdm2 (a.a. 801-1050) and p53 (a.a. 880-1050).
• The catalytic domain (a.a. 208-560) showed significantly reduced proteolytic activity compared to full-length or truncated USP7.

Conclusions:

• The N-terminal domain is not essential for USP7-mediated deubiquitination of mdm2 and p53.
• The C-terminal domain of USP7 is critical for maintaining catalytic activity, substrate binding, and inhibitor interaction.
• USP7's C-terminal region likely stabilizes the enzyme's active conformation.