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

Abstract

USP7, also known as the hepes simplex virus associated ubiquitin-specific protease (HAUSP), deubiquitinates both mdm2 and p53, and plays an important role in regulating the level and activity of p53. Here, we report that deletion of the TRAF-like domain at the N-terminus of USP7, previously reported to contain the mdm2/p53 binding site, has no effect on USP7 mediated deubiquitination of Ub n-mdm2 and Ub n-p53. Amino acids 208–1102 were identified to be the minimal length of USP7 that retains proteolytic activity, similar to full length enzyme, towards not only a truncated model substrate Ub-AFC, but also Ub n-mdm2, Ub n-p53. In contrast, the catalytic domain of USP7 (amino acids 208–560) has 50–700 fold less proteolytic activity towards different substrates. Moreover, inhibition of the catalytic domain of USP7 by Ubal is also different from the full length or TRAF-like domain deleted proteins. Using glutathione pull-down methods, we demonstrate that the C-terminal domain of USP7 contains additional binding sites, a.a. 801–1050 and a.a. 880–1050 for mdm2 and p53, respectively. The additional USP7 binding site on mdm2 is mapped to be the C-terminal RING finger domain (a.a. 425–491). We propose that the C-terminal domain of USP7 is responsible for maintaining the active conformation for catalysis and inhibitor binding, and contains the prime side of the proteolytic active site.