Enabling Protein Degradation Drug Discovery

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  • Name
    Catalogue Number
    Size
    Price
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  • Name:
    UBE2K (UbcH1) [untagged]
    Catalogue Number:
    62-0039-020
    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
    ~23 kDa
  • Stability
    12 months at -70°C; aliquot as required
  • Protein Sequence
    Accession number: NP_005330. 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 UBE2K was validated by loading E1 UBE1 activated ubiquitin onto the active cysteine of the UBE2K E2 enzyme via a transthiolation reaction. Incubation of the UBE1 and UBE2K 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/UBE2K 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). UBE2K is a member of the E2 conjugating enzyme family and cloning of the human gene was first described by Kalchman et al. (1996). Human UBE2K shares 100% amino acid identity with bovine UBE2K and significant homology with yeast UBE2K, UBE2D2 and UBE2D1 (Kalchman et al., 1996). Interaction and selective binding of UBE2K to the N-terminus of huntingtin, the causal gene product in Huntington’s disease has been demonstrated (Kalchman et al.,1996). In a yeast 2 hybrid screen binding of the RING finger protein RNF2 to UBE2K has also been determined (Christensen et al., 2007; Lee et al., 2001). UBE2K binds directly to the BRCA1 RING motif and is active with BRCA1-BARD1 in in vitro autoubiquitylation assays (Christensen et al., 2007). UBE2K directs the synthesis of Lys63- or Lys48-linked ubiquitin chains on BRCA1. UBE2K has been found to synthesize in vitro unanchored Lys(48)-linked poly-ubiquitin chains from mono- or poly-ubiquitin, E1, and ATP; thus, UBE2K has distinct binding sites for donor and acceptor (poly)Ub (Yao and Cohen, 2000). UBE2K was identified in a screen for novel SUMO targets, however attachment of SUMO to UBE2K in vitro severely impairs ubiquitin thioester and unanchored ubiquitin chain formation (Pichler et al., 2005). The ubiquitin-proteasome system malfunction in Alzheimer’s disease (AD) has been attributed to neurotoxicity and proteasome inhibition by Abeta, which is mediated by an increase in the levels of UBE2K found in the brains of patients with AD. UBE2K’s contribution to neurotoxicity is mediated by a ubiquitin B mutant (UBB+1), a potent inhibitor of proteasomes found in patients with AD (Song and Jung, 2004).

References:

Kalchman MA, Graham RK, Xia G, Koide B, Hodgson JG, Graham KC, Goldberg YP, Gietz RD, Pickart CM, Haydan MR (1996) Huntingtin is ubiquitinated and interacts with a specific ubiquitin-conjugating enzyme. J Biol Chem 271, 19385-94.
Lee SJ, Choi JY, Sung YM, Park H, Rhim H, Kang S (2001) E3 ligase activity of RING finger proteins that interact with Hip-2, a human ubiquitin-conjugating enzyme. FEBS Lett 503, 61-4.
Pichler A, Knipscheer P, Oberhofer E, van Dijk WJ, Korner R, Olsen JV, Jentsch S, Melchior F, Sixma TK (2005) SUMO modification of the ubiquitin-conjugating enzyme E2-25K. Nat Struct Mol Biol 12, 264-9.
Song S, Jung YK (2004) Alzheimer’s disease meets the ubiquitin-proteasome system. Trends Mol Med 10, 565-70.
Yao and Cohen (2000) Cyclization of polyubiquitin by the E2-25K ubiquitin conjugating enzyme. J Biol Chem 275, 36862-8.