Enabling Protein Degradation Drug Discovery

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  • Name
    Catalogue Number
    Size
    Price
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  • Name:
    UBE2A (HR6A) [6His-tagged]
    Catalogue Number:
    62-0071-020
    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
    ~21 kDa
  • Stability
    12 months at -70°C; aliquot as required
  • Protein Sequence
    Accession number: NP_003327. 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-UBE2A was validated by loading E1 UBE1 activated ubiquitin onto the active cysteine of the His-UBE2A E2 enzyme via a transthiolation reaction. Incubation of the UBE1 and His-UBE2A 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-UBE2A 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 the 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). UBE2A is a member of the E2 conjugating enzyme family and cloning of the human gene was first described by Koken et al. (1991). UBE2A shares 70% identity with its yeast homologue but lacks the acidic C-terminal domain. The ring finger proteins RAD5 and RAD18 interact with UBE2A and other members of the RAD6 pathway (Ulrich and Jentsch, 2000). Phosphorylation of UBE2A by CDK1 and 2 increases its activity during the G2/M phase of the cell cycle (Sarcevic et al., 2002). UBE2A is required for post-replicative DNA damage repair in eukaryotic cells and it is thought binding to ZNF198 may be involved in this process (Kunapuli et al., 2003). A nonsense mutation resulting in the loss of a 25 amino acid region in the C-terminal domain of UBE2A has been identified as a cause of a novel X-linked mental retardation (XLMR) syndrome (Nascimento et al., 2006).

References:

Koken MH, Reynolds P, Jaspers-Dekker I, Prakash L, Prakash S, Bootsma D, Hoeijmakers JH (1991) Structural and functional conservation of two human homologs of the yeast DNA repair gene RAD6. Proc Natl Acad Sci U S A 88, 8865-9.
Kunapuli P, Somerville R, Still IH, Cowell JK (2003) ZNF198 protein, involved in rearrangement in myeloproliferative disease, forms complexes with the DNA repair-associated HHR6A/6B and RAD18 proteins. Oncogene 22, 3417-23.
Nascimento RM, Otto PA, de Brouwer AP, Vianna-Morgante AM (2006) UBE2A, which encodes a ubiquitin-conjugating enzyme, is mutated in a novel X-linked mental retardation syndrome. Am J Hum Genet 79, 549-55.
Sarcevic B, Mawson A, Baker RT, Sutherland RL (2002) Regulation of the ubiquitin-conjugating enzyme hHR6A by CDK-mediated phosphorylation. EMBO J 21, 2009-18.
Ulrich HD, Jentsch S (2000) Two RING finger proteins mediate cooperation between ubiquitin-conjugating enzymes in DNA repair. EMBO J 19, 3388-97.