Linking Ubiquitin Research to Drug Discovery

*Purity of all products >95% unless otherwise stated
  • Name
    Catalogue Number
    Size
    Price
    Add to Basket
  • Name:
    USP14 [6His-tagged]
    Catalogue Number:
    64-0018-050
    Size:
    50 µg
    Price:
    £195
    Add To Basket
  • Species
    human
  • Source
    E. coli
  • Quantity
    50 µg
  • Storage
    -70°C
  • Concentration
    0.5mg/ml
  • Formulation
    50mM HEPES pH7.5, 150mM NaCl2, 2mM DTT, 10% Glycerol
  • Molecular Weight
    ~58.5kDa
  • Stability
    12 months at -70°C; aliquot as required
  • Protein Sequence
    Accession number: AAH03556. For full protein sequence information download the Certificate of Analysis pdf.
  • QA; Protein Identification
    Confirmed by mass spectrometry.
  • QA; Activity
    Deubiquitylase Enzyme Assay: His-USP14 has limited activity against the fluorogenic substrate Ubiquitin-Rhodamine110-Glycine. The DUB activity of His-USP14 is known to be potentiated by proteasomes. See Cat#64-1010-096 for the dual product (USP14 & 26S proteasome [Ub-VS]) proteasome-activated USP14.

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 specific protease 14 (USP14) is a member of the cysteine protease enzyme family and cloning of the gene was first described by Deshpande et al. (1996). 

USP14 is a proteasome–associated DUB enzyme. Mammalian proteasomes are associated with three DUBs: USP14, UCHL5 (UCH37) and RPN11 (POH1). UCHL5 and USP14 reside on the 19S regulatory particle and remove ubiquitin from the substrate before substrate degradation whereas RPN11’s activity is delayed until the proteasome is committed to degrading the substrate (Lee et al., 2010). Ubiquitin-tagged substrates are degraded by the 26S proteasome, which is a multi-subunit complex comprising a proteolytic 20S core particle capped by 19S regulatory particles (Wang et al., 2007). The approval of bortezomib for the treatment of multiple myeloma validated the 20S core particle as an anticancer drug target. Recent experiments have now shown that the 19S regulatory particle is also a potential anticancer drug target (D’Arcy et al., 2011). Alterations in USP14 can lead to proteasome dysfunction and neurological disease. Recent in vitro experiments show that USP14 can also stabilize the expression of over-expressed, disease-associated proteins such as tau and ataxin-3 (Jin et al., 2012).  A number of other studies over the years have implicated USP14 in cancer. Ishiwata et al., (2001) first found that the USP14 expression was upregulated in leukemic cells, Shinji et al., (2006) found that the USP14 expression in colorectal cancer is associated with liver and lymph node metastases and Chuensumran, et al., (2011) that USP14 expression is associated with intrahepatic cholangiocarcinoma cell differentiation. A recent review by Wu et al. (2013) also reports that USP14 is a tumour-promoting factor and a promising therapeutic target for non-small cell lung cancer (NSCLC). 

The DUB activity of USP14 is known to be activated by proteasomes and alone USP14 has little DUB activity and as such may be used as a control in experiments alongside those performed with proteasome-activated USP14: Such experiments are typically performed using a proteasome preparation where the native DUBs – including USP14 – have been removed and/or inactivated before the proteasome preparation is added back to recombinantly expressed USP14.

References:

Chuensumran U, Saelee P, Punyarit P, Wongkham S, Pairojkul C, Chauin S, et al. (2011)  Ubiquitin-specific protease 14 expression associated with intrahepatic cholangiocarcinoma cell differentiation. Asian Pac J Cancer Prev 12, 775-779.
D’Arcy P, Brnjic S, Olofsson MH, Fryknas M, Lindsten K, De Cesare M, et al. (2011) Inhibition of  proteasome deubiquitinating activity as a new cancer therapy. Nature Medicine 17, 1636-1640.
Deshpande KL, Seubert PH, Tillman DM, Farkas WR and Katze JR (1996) Cloning and characterization of cDNA encoding the rabbit tRNA-guanine transglycosylase 60-kilodalton subunit. Arch Biochem Biophys 326, 1-7.
Ishiwata S, Katayama J, Shindo H, Ozawa Y, Itoh K and Mizugaki M (2001) Increased expression of queuosine synthesizing enzyme, tRNA-guanine transglycosylase, and queuosine levels in tRNA of leukemic cells. J Biochem 129, 13-17.
Jin YN, Chen PC, Watson JA, Walters BJ, Phillips SE, Green K, et al. (2012) Usp14 deficiency increases tau phosphorylation without altering tau degradation or causing tau-dependent deficits. PloS one 7, e47884.
Komander D, Clague MJ and Urbe S (2009) Breaking the chains: structure and function of the deubiquitinases. Nat Rev Mol Cell Biol 10, 550-563.
Lee BH, Lee MJ, Park S, Oh DC, Elsasser S, Chen PC, et al. (2010) Enhancement of proteasome activity by a small molecule inhibitor of USP14. Nature 467, 179-184.
Reyes-Turcu FE, Ventii KH and Wilkinson KD (2009) Regulation and cellular roles of ubiquitin-specific deubiquitinating enzymes.Ann Rev Biochem 78, 363-397.
Shinji S, Naito Z, Ishiwata S, Ishiwata T, Tanaka N, Furukawa K, et al. (2006) Ubiquitin-specific protease 14 expression in colorectal cancer is associated with liver and lymph node metastases. Oncol Rep 15, 539-543.
Wang X, Chen CF, Baker PR, Chen PL, Kaiser P and Huang L (2007) Mass spectrometric characterization of the affinity-purified human 26S proteasome complex. Biochemistry 46, 3553-3565.
Wu N, Liu C, Bai C, Han YP, Cho WC and Li Q (2013) Overexpression of Deubiquitinating Enzyme USP14 in Lung Adenocarcinoma Promotes Proliferation through the Accumulation of beta-Catenin. Int J Mol Sci 14, 10749-10760.