Enabling Protein Degradation Drug Discovery

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  • Name
    Catalogue Number
    Size
    Price
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  • Name:
    UBE2N (UBC13) [6His-tagged]
    Catalogue Number:
    62-0045-020
    Size:
    20 µg
    Price:
    £100
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  • Species
    Human
  • Source
    Sf21 insect cell-baculovirus expression
  • Quantity
    20 µg
  • Storage
    -70°C
  • Concentration
    1 mg/ml
  • Formulation
    50 mM HEPES pH 7.5, 150 mM sodium chloride, 2 mM dithiothreitol, 10% glycerol
  • Molecular Weight
    ~20 kDa
  • Stability
    12 months at -70°C; aliquot as required
  • Protein Sequence
    Accession number: NP_003339. For full protein sequence information download the Certificate of Analysis pdf.
  • QA; Protein Identification
    Confirmed by mass spectrometry.
  • QA; Activity
    E2-Ubiquitin Thioester Loading Assay: The activity of His-UBE2N was validated by loading E1 UBE1 activated ubiquitin onto the active cysteine of the His-UBE2N E2 enzyme via a transthiolation reaction. Incubation of the UBE1 and His-UBE2N enzymes in the presence of ubiquitin and ATP at 30°C was compared at two time points, T0 and T10 minutes. Sensitivity of the ubiquitin/His-UBE2N thioester bond to the reducing agent DTT was confirmed.

The enzymes of the ubiquitylation pathway play a pivotal role in a number of cellular processes including regulated and targeted proteosomal degradation of substrate proteins. Three classes of enzymes are involved in the process of ubiquitylation; activating enzymes (E1s), conjugating enzymes (E2s) and protein ligases (E3s). UBE2N is a member of the E2 conjugating enzyme family and cloning of the human gene was first described by Yamaguchi et al. (1996). The human UBE2N sequence shares 80% identity with the Drosophila ‘bendless’ gene product. In yeast, UBE2N forms a specific heteromeric complex with MMS2 a signalling component of the RAD6 pathway. The RAD6 pathway is central to DNA repair and two major components of this pathway are RAD6 and the MMS2/UBE2N heterodimer which are recruited to chromatin by the RING finger proteins RAD18 and RAD5, respectively (Hofmann and Pickart, 1999). Proliferating Cell Nuclear Antigen (PCNA) is modified by Lys-63-linked polyubiquitylation, which requires MMS2, UBE2N and RAD5. Depletion of UBE2N in vitro results in severe growth defects caused by chromosome instability, as well as hypersensitivity to UV and ionizing radiation, this is consistent with a conserved role for UBE2N in RAD6/RAD18-dependent post-replication repair (Zhao et al., 2007). Cytokine receptor signalling results in complex formation of protein kinases such as CD40 with TRAF2 and TRAF3, UBE2N, cellular inhibitor of apoptosis protein-1 (CIAP1) and -2 (CIAP2), IKK-α and MEKK1. Translocation of a TRAF2, UBE2N, and IKK-α complex from the membrane to the cytosol is initiated by a CIAP1/CIAP2-induced degradation of TRAF3 which results in activation of MEKK1 and MAP kinase cascades (Matsuzawa et al., 2008). Heterozygous UBE2N mice exhibit selectively impaired activation of signal transduction pathways initiated by TNFr and show reduced ubiquitylation of TRAF6.  Reducing UBE2N activity may have therapeutic uses in controlling inflammatory responses. (Matsuzawa et al., 2008)

References:

Hofmann RM, Pickart CM (1999) Noncanonical MMS2-encoded ubiquitin-conjugating enzyme functions in assembly of novel polyubiquitin chains for DNA repair. Cell 96, 645-53.

Matsuzawa A, Tseng PH, Vallabhapurapu S, Luo JL, Zhang W, Wang H, Vignali DA, Gallagher E, Karin M (2008) Essential cytoplasmic translocation of a cytokine receptor-assembled signaling complex. Science 321, 663-8.

Yamaguchi T, Kim NS, Sekine S, Seino H, Osaka F, Yamao F, Kato S (1996) Cloning and expression of cDNA encoding a human ubiquitin-conjugating enzyme similar to the Drosophila bendless gene product. J Biochem 120, 494-97.

Zhao GY, Sonoda E, et al. (2007) A critical role for the ubiquitin-conjugating enzyme Ubc13 in initiating homologous recombination. Mol Cell 25, 663-75.