Enabling Protein Degradation Drug Discovery

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  • Name
    Catalogue Number
    Size
    Price
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  • Name:
    HUWE1 [GST-tagged]
    Catalogue Number:
    63-0042-025
    Size:
    25 µg
    Price:
    £250
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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
    ~97kDa
  • Stability
    12 months at -70°C; aliquot as required
  • Protein Sequence
    Accession number: NP_113584.3. 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-HUWE1 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-HUWE1 for 60 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-HUWE1 (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-HUWE1.

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). Hect, Uba, and Wwe Domains containing protein 1 (HUWE1) is a member of the Homologous to the E6AP Carboxyl Terminus (HECT) E3 protein ligase family and cloning of the human gene was first described by (Liu et al., 2005). HUWE1 ubiquitylates the Histone proteins, H1, H2A, H2B, H3 and H4 (Liu et al., 2005). PCR analysis of X chromosome-specific array comparative genomic hybridization has identified over expression of HUWE1 in the blood of individuals affected by nonsyndromic X-linked mental retardation (Froyen et al., 2008). HUWE1 catalyses K48 linked poly-ubiquitylation and proteasomal degradation of the transcription factor Miz1 (Myc-Interacting Zinc finger protein 1) (Yang et al., 2010). HUWE1 has also been shown to ubiquitylate Mcl1, p53, MyoD and cMyc (Zhong et al., 2005; Chen et al., 2005; Noy et al., 2012; Inoue et al., 2013). HUWE1 null mice display increased number and severity of skin tumors which can be reversed by concomitant genetic knockout of c-Myc (Inoue et al., 2013).

References:
Chen D, Kon N, Li M, Zhang W, Qin J, Gu W. (2005) ARF-BP1/Mule is a critical mediator of the ARF tumor suppressor. Cell 121, 1071–1083.

Froyen G, Corbett M, Vandewalle J, Jarvela I, Lawrence O, Meldrum C, Bauters M, Govaerts K, Vandeleur L, Van Esch H, Chelly J, Sanlaville D, et al. (2008) Submicroscopic duplications of the hydroxysteroid dehydrogenase HSD17B10 and the E3 ubiquitin ligase HUWE1 are associated with mental retardation. Am J Hum Genet 82, 432-443.

Liu Z, Oughtred R, Wing SS. (2005) Characterization of E3-Histone, a novel testis ubiquitin protein ligase which ubiquitinates histones. Molec Cell Biol 25, 2819-2831.

Inoue S, Hao Z, Elia AJ, Cescon D, Zhou L, et al. (2013) Mule/Huwe1/Arf-BP1 suppresses Ras-driven tumorigenesis by preventing c-Myc/Miz1-mediated down-regulation of p21 and p15. Genes Dev 27, 1101-14.

Noy T, Suad O, Taglicht D, Ciechanover A. (2012) HUWE1 ubiquitinates MyoD and targets it for proteasomal degradation. Biochem Biophys Res Commun 418, 408-13.

Yang Y, Do H, Tian X, Zhang C, Liu X, Dada LA, Sznajder JI, Liu J. (2010) E3 ubiquitin ligase Mule ubiquitinates Miz1 and is required for TNFalpha-induced JNK activation. PNAS 27, 13444-9.