Linking Ubiquitin Research to Drug Discovery

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  • Name
    Catalogue Number
    Size
    Price
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  • Name:
    USP5 [6His-tagged]
    Catalogue Number:
    64-0002-050
    Size:
    50 µg
    Price:
    £195
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  • Species
    Human
  • Source
    E. coli expression
  • Quantity
    50 µg
  • Storage
    -70°C
  • Concentration
    0.5 mg/ml
  • Formulation
    50 mM HEPES pH 7.5, 150 mM sodium chloride, 2 mM dithiothreitol, 10% glycerol
  • Molecular Weight
    ~98 kDa
  • Stability
    12 months at -70°C; aliquot as required
  • Protein Sequence
    Accession number: P45974. For full protein sequence information download the Certificate of Analysis pdf.
  • QA; Protein Identification
    Confirmed by mass spectrometry.
  • QA; Activity
    Deubiquitylating Enzyme Assay: The activity of His-USP5 was validated by determining the increase in fluorescence measured as a result of the enzyme catalysed cleavage of the fluorogenic substrate Ubiquitin-Rhodamine110-Glycine generating Ubiquitin and Rhodamine110-Glycine. Incubation of the substrate in the presence or absence of His-USP5 was compared confirming the deubiquitylating activity of His-USP5.

The Deubiquitylating enzymes (DUBs) regulate ubiquitin dependent signaling pathways. The activities of the DUBs include the generation of free ubiquitin from precursor molecules, the recycling of ubiquitin following substrate degradation to maintain cellular ubiquitin homeostasis and the removal of ubiquitin or ubiquitin-like protein (UBL) modifications through chain editing to rescue proteins from proteasomal degradation or to influence cell signalling events (Komander et al., 2009). There are two main classes of DUB, cysteine proteases and metalloproteases. Ubiquitin specific processing protease 5 (USP5) is a member of the cysteine protease enzyme family and cloning of the human gene was first described by Wilkinson et al. (1995). USP5 protein contains an N-terminal zinc finger ubiquitin-binding domain (ZNF-UBP), a ubiquitin-specific processing protease (UBP) domain containing the active-site cys and his boxes, and two ubiquitin-associated domains (UBA1 and UBA2) (Reyes-Turcu et al., 2006). Crystal structures of the ZNF-UBP domain have revealed a deep binding pocket where the C-terminal diglycine motif of ubiquitin is inserted explaining the specificity of USP for an unmodified C-terminus on the proximal subunit of polyubiquitin (Reyes-Turcu et al., 2006). Recently USP5 has been identified in association with the 26S proteasome alongside other proteins known in complex as the UBL interactome (Besche et al., 2009).

References:

Besche H, Haas W, Gygi S, Goldberg A (2009) Isolation of mammalian 26S proteasomes and p97/VCP complexes using the ubiquitin-like domain from HHR23B reveals novel proteasome-associated proteins. Biochemistry.48, 2538-49.

Komander D, Clague MJ, Urbe S (2009) Breaking the chains: structure and function of the deubiquitinases. Nat Rev Mol Cell Biol 10, 550-63.

Reyes-Turcu FE, Horton JR, Mullally JE, Heroux A, Cheng X, Wilkinson KD (2006) The ubiquitin binding domain ZnF UBP recognizes the C-terminal diglycine motif of unanchored ubiquitin. Cell 124, 1197-208.

Wilkinson KD (1995) Roles of ubiquitinylation in proteolysis and cellular regulation. Annu Rev Nutr 15, 161-89.