title: 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.
authors.affiliation: GlaxoSmithKline, King of Prussia, PA 19406, USA. jane.2.ma@gsk.com
authors.forename: Jianhong
authors.initials: J
authors.lastname: Ma
authors.forename: John D
authors.initials: JD
authors.lastname: Martin
authors.forename: Yu
authors.initials: Y
authors.lastname: Xue
authors.forename: Leng A
authors.initials: LA
authors.lastname: Lor
authors.forename: Karen M
authors.initials: KM
authors.lastname: Kennedy-Wilson
authors.forename: Robert H
authors.initials: RH
authors.lastname: Sinnamon
authors.forename: Thau F
authors.initials: TF
authors.lastname: Ho
authors.forename: Guofeng
authors.initials: G
authors.lastname: Zhang
authors.forename: Benjamin
authors.initials: B
authors.lastname: Schwartz
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Tummino
authors.forename: Zhihong
authors.initials: Z
authors.lastname: Lai
mesh_terms: D020816:Amino Acid Motifs / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics; D016158:Genes, p53; D006801:Humans; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D051736:Proto-Oncogene Proteins c-mdm2 / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D043222:Ubiquitin Thiolesterase / Q000737:chemistry*; D000076084:Ubiquitin-Specific Peptidase 7; D054875:Ubiquitination
chemical_list: D011993:Recombinant Fusion Proteins; D016159:Tumor Suppressor Protein p53; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2; C464569:USP7 protein, human; D043222:Ubiquitin Thiolesterase; D000076084:Ubiquitin-Specific Peptidase 7