Biochemical characterization of USP7 reveals post-translational modification sites and structural requirements for substrate processing and subcellular localization.
Ubiquitin specific protease 7 (USP7) belongs to the family of deubiquitinating enzymes. Among other functions, USP7 is involved in the regulation of stress response pathways, epigenetic silencing and the progress of infections by DNA viruses. USP7 is a 130-kDa protein with a cysteine peptidase core, N- and C-terminal domains required for protein-protein interactions. In the present study, recombinant USP7 full length, along with several variants corresponding to domain deletions, were expressed in different hosts in order to analyze post-translational modifications, oligomerization state, enzymatic properties and subcellular localization patterns of the enzyme. USP7 is phosphorylated at S18 and S963, and ubiquitinated at K869 in mammalian cells. In in vitro activity assays, N- and C-terminal truncations affected the catalytic efficiency of the enzyme different. Both the protease core alone and in combination with the N-terminal domain are over 100-fold less active than the full length enzyme, whereas a construct including the C-terminal region displays a rather small decrease in catalytic efficiency. Limited proteolysis experiments revealed that USP7 variants containing the C-terminal domain interact more tightly with ubiquitin. Besides playing an important role in substrate recognition and processing, this region might be involved in enzyme dimerization. USP7 constructs lacking the N-terminal domain failed to localize in the cell nucleus, but no nuclear localization signal could be mapped within the enzyme's first 70 amino acids. Instead, the tumor necrosis factor receptor associated factor-like region (amino acids 70-205) was sufficient to achieve the nuclear localization of the enzyme, suggesting that interaction partners might be required for USP7 nuclear import.
Fernández-Montalván
Amaury
A
Expertise Platform Proteases, Novartis Institutes for Biomedical Research, Basel, Switzerland. amaury.fernandez@merckserono.net
Bouwmeester
Tewis
T
Joberty
Gerard
G
Mader
Robert
R
Mahnke
Marion
M
Pierrat
Benoit
B
Schlaeppi
Jean-Marc
JM
Worpenberg
Susanne
S
Gerhartz
Bernd
B
D000595:Amino Acid Sequence; D001665:Binding Sites; D020134:Catalytic Domain / Q000235:genetics; D002460:Cell Line; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism*; D002850:Chromatography, Gel; D003600:Cytosol / Q000378:metabolism*; D019281:Dimerization; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D013058:Mass Spectrometry; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014162:Transfection; D043222:Ubiquitin Thiolesterase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000076084:Ubiquitin-Specific Peptidase 7
D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C464569:USP7 protein, human; D043222:Ubiquitin Thiolesterase; D000076084:Ubiquitin-Specific Peptidase 7