Enabling Protein Degradation Drug Discovery

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  • Name
    Catalogue Number
    Size
    Price
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  • Name:
    ZNRF2 [GST-tagged]
    Catalogue Number:
    63-0041-025
    Size:
    25 µg
    Price:
    £325
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  • Species
    human
  • Source
    E.coli
  • Quantity
    25 µg
  • Storage
    -70°C
  • Concentration
    0.5mg/ml
  • Formulation
    50 mM HEPES pH 7.5, 150 mM sodium chloride, 2 mM dithiothreitol, 10% glycerol
  • Molecular Weight
    ~50kDa
  • Stability
    12 months at -70°C; aliquot as required
  • Protein Sequence
    Accession number: Q8NHG8. For full protein sequence information download the Certificate of Analysis pdf.
  • QA; Protein Identification
    Confirmed by mass spectrometry.
  • QA; Activity
    E3 Ligase Assay: The ubiquitin conjugating activity of GST-ZNRF2 was validated through its ability to catalyse the generation of polyubiquitin chains in the presence of the E1 activating enzyme His-UBE1, the E2 conjugating enzyme His-UBE2D3 (UbcH5c) (several E2s were tested, data generated with this E2 is provided by way of example) and ubiquitin. Incubation of GST-ZNRF2 for 30 minutes at 30oC in the presence of ubiquitin, His-UBE1, His-UBE2D3 and ATP (Lane 1) was compared alongside two control reactions with either ATP (Lane 2) or GST-ZNRF2 (Lane 3) excluded from the reaction. Ubiquitin conjugates were identified by Western blotting using an anti-ubiquitin conjugate antibody and these were observed only in the presence of both ATP and GST-ZNRF2.

The enzymes of the ubiquitylation pathway play a pivotal role in a number of cellular processes including the regulated and targeted proteasome-dependent 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). Zinc and Ring Finger protein 2 (ZNRF2) is a member of the Really Interesting New Gene (RING) E3 protein ligase family and cloning of the human gene was first described by Araki et al. (2001). ZNRF2 has been shown to be expressed in both the central and peripheral nervous system of rats during development and in adulthood (Araki et al., 2001). Ube2D2 and Ube2D3 have been identified as supporting E2 conjugating enzymes in the in vitro ubiquitylation activity of ZNRF2 and mutation of the Zinc finger Ring finger domain of ZNRF2 showed that it was required for E3 ligase activity of ZNRF2 (Araki and Milbrandt 2003). ZNRF2 also regulates non-canonical ubiquitylation by binding to heterodimers of Ube2N/Uev1a via its RING domain and recruiting additional E2 conjugating enzymes which allow for K48 linked poly-ubiquitylation to occur as well as K63 linked polyubiquitylation (Plans et al., 2006).

References:
Araki T, Nagarajan R, Milbrandt J. (2001) Identification of genes induced in peripheral nerve after injury: expression profiling and novel gene discovery. J Biol. Chem 276, 34131-34141.

Araki T, Milbrandt J. (2003) ZNRF proteins constitute a family of presynaptic E3 ubiquitin ligases J. Neurosci 23, 9385-9394.

Plans V, Scheper J, Soler M, Loukili N, Okano Y, Thomson TM. (2006) The RING finger protein RNF8 recruits UBC13 for lysine 63-based self polyubiquitylation. J Cell Biochem 97, 572-82.