Linking Ubiquitin Research to Drug Discovery

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  • Name
    Catalogue Number
    Size
    Price
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  • Name:
    UCHL3 [GST-tagged]
    Catalogue Number:
    64-0027-050
    Size:
    50 µg
    Price:
    £195
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  • Species
    human
  • Source
    E.coli
  • 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
    ~53 kDa
  • Stability
    12 months at -70°C; aliquot as required
  • Protein Sequence
    Accession number: AAH18125. For full protein sequence information download the Certificate of Analysis pdf.
  • QA; Protein Identification
    Confirmed by mass spectrometry.
  • QA; Activity
    Deubiquitylase Enzyme Assay: The activity of GST-UCHL3 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 GST-UCHL3 was compared confirming the deubiquitylating activity of GST-UCHL3.

Deconjugating enzymes (DCEs) are proteases that process ubiquitin or ubiquitin-like gene products, reverse the modification of proteins by a single ubiquitin or ubiquitin-like protein (UBL) and remodel polyubiquitin (or poly-UBL) chains on target proteins (Reyes-Turcu et al., 2009). The deubiquitylating – or deubiquitinating – enzymes (DUBs) represent the largest family of DCEs and regulate ubiquitin dependent signalling 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 proteins (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 carboxyl-terminal hydrolase L3 (UCHL3) is a member of the cysteine protease enzyme family and cloning of the human gene was first described by Wilkinson et al. (1989). The UCH subfamily of DUBs consists of four members: UCHL1, UCHL3, UCHL5 and BRCA1-associated protein-1 (BAP1) with UCHL3 sharing 54% homology with UCHL1 (Day and Thompson, 2010). Unlike other UCHs, UCHL3 can function as a C-terminal hydrolase for both NEDD8 and ubiquitin. UCHL3 may play a physiologically significant role in the cleavage of the C-terminus of NEDD8, which is required for NEDD8 to conjugate to target proteins (Wada et al., 1998).  There is accumulating evidence suggesting that the expression and activity of UCH enzymes in cancers are abnormal. Increased levels of both UCHL1 and UCHL3 mRNA are associated with early tumour recurrence of invasive breast cancer and poor prognosis (Fang et al., 2010).
References:

Day IN, Thompson RJ (2010) UCHL1 (PGP 9.5): neuronal biomarker and ubiquitin system protein. Prog Neurobiol 90, 327-362.

Fang Y, Fu D, Shen XZ (2010) The potential role of ubiquitin c-terminal hydrolases in oncogenesis. Biochim Biophys Acta 1806, 1-6.

Komander D, Clague MJ, Urbe S (2009) Breaking the chains: structure and function of the deubiquitinases. Nat Rev Mol Cell Biol 10, 550-563.

Reyes-Turcu FE, Ventii KH, Wilkinson KD (2009) Regulation and cellular roles of ubiquitin-specific deubiquitinating enzymes. Ann Rev Biochem 78, 363-397.

Wada H, Kito K, Caskey LS, Yeh ET, Kamitani T (1998) Cleavage of the C-terminus of NEDD8 by UCH-L3. Biochem Biophys Res Commun 251, 688-692.

Wilkinson KD, Lee KM, Deshpande S, Duerksen-Hughes P, Boss JM, Pohl J (1989) The neuron-specific protein PGP 9.5 is a ubiquitin carboxyl-terminal hydrolase. Science 246, 670-673.