Enabling Protein Degradation Drug Discovery

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  • Name
    Catalogue Number
    Size
    Price
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  • Name:
    UBE2E3 (UbcH9) [6His-tagged]
    Catalogue Number:
    62-0076-020
    Size:
    20 µg
    Price:
    £100
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  • Species
    Human
  • Source
    E. coli 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
    ~26 kDa
  • Stability
    12 months at -70°C; aliquot as required
  • Protein Sequence
    Accession number: NP_006348. 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-UBE2E3 was validated by loading E1 UBE1 activated ubiquitin onto the active cysteine of the His-UBE2E3 E2 enzyme via a transthiolation reaction. Incubation of the UBE1 and His-UBE2E3 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-UBE2E3 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 proteasomal 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). UBE2E3 is a member of the E2 ubiquitin-conjugating enzyme family and cloning of the gene was first described by Ito et al., (1999). UBE2E3 binds to the RING-finger proteins ARA54 and RNF8, thought to act as E3 ligases in the ubiquitylation of nuclear proteins (Ito et al., 2001). The epithelial Na+ channel (ENaC) is regulated by UBE2E3 and the E3 ligase NEDD4.2. UBE2E3 interacts with NEDD4.2 via its UBC domain and ubiquitylation of ENaC occurs by NEDD4.2 binding the PY motifs of its α, β and γ subunits (Debonneville and Staub. 2004). NEDD4.2 is a negative regulator of ENaC and deletions in the PY motifs of the β and γ subunits of ENaC cause Liddle’s syndrome, an inherited form of hypertension. The loss of NEDD4.2 binding sites in mutated ENaC causes an increase in channel number at the cell surface and increased Na+ reabsorption by the distal nephron, resulting in hypertension (Abriel et al., 1999).

References:

Abriel H, Loffing J, Rebhun JF, Pratt JH, Schild L, Horisberger JD, Rotin D, Staub O (1999) Defective regulation of the epithelial Na+ channel by Nedd4 in Liddle’s syndrome. J Clin Invest 103, 667-73.

Debonneville C, Staub O (2004) Participation of the ubiquitin-conjugating enzyme UBE2E3 in Nedd4-2-dependent regulation of the epithelial Na+ channel. Mol Cell Biol 24, 2397-409.

Ito K, Adachi S, Iwakami R, Yasuda H, Muto Y, Seki N, Okano Y (2001) N-Terminally extended human ubiquitin-conjugating enzymes (E2s) mediate the ubiquitination of RING-finger proteins, ARA54 and RNF8. Eur J Biochem 268, 2725-32.

Ito K, Kato S, Matsuda Y, Kimura M, Okano Y (1999) cDNA cloning, characterization, and chromosome mapping of UBE2E3 (alias UbcH9), encoding an N-terminally extended human ubiquitin-conjugating enzyme. Cytogenet Cell Genet 84, 99-104.