Enabling Protein Degradation Drug Discovery

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  • Name
    Catalogue Number
    Size
    Price
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  • Name:
    UBE2T (HSPC150) [6His-tagged]
    Catalogue Number:
    62-0057-020
    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
    ~25 kDa
  • Stability
    12 months at -70°C; aliquot as required
  • Protein Sequence
    Accession number: NP_054895. 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-UBE2T was validated by loading E1 UBE1 activated ubiquitin onto the active cysteine of the His-UBE2T E2 enzyme via a transthiolation reaction. Incubation of the UBE1 and His-UBE2T 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-UBE2T 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 proteosomal 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). UBE2T is a member of the E2 conjugating enzyme family and cloning of the human gene was first described by Zhang et al. (2000). UBE2T is integral to the Fanconi Anemia (FA) pathway for DNA damage repair. UBE2T binds to the C-terminal PH domain of FANCL the ubiquitin ligase subunit of the FA core complex, which leads to the monoubiquitylation of FANCD2 and FANCI (Longerich et al., 2009; Machida et al., 2006). E3 ligase activity is not determined by assembly of the FA core complex but by the DNA damage-induced subcellular localization of the complex to chromatin. UBE2T and FANCD2 access this subcellular fraction independently and FANCD2 monoubiquitylation is regulated by the formation of an E2/E3 holoenzyme on chromatin. DNA damage in UBE2T-depleted human osteosarcoma cells leads to the formation of abnormal chromosomes that are a hallmark of FA (Alpi et al., 2007). UBE2T expression has been analysed in lung cancer tissue and compared to normal human tissue. UBE2T was found to be significantly upregulated at both the protein and mRNA level suggesting involvement in the malignant cell phenotype (Hao et al., 2008).

References:

Alpi A, Langevin F, Mosedale G, Machida YJ, Dutta A, Patel KJ (2007) UBE2T, the Fanconi anemia core complex, and FANCD2 are recruited independently to chromatin: a basis for the regulation of FANCD2 monoubiquitination. Mol Cell Biol 27, 8421-30.

Hao J, Xu A, Xie X, Tian T, Gao S, Xiao X, He D (2008) Elevated expression of UBE2T in lung cancer tumors and cell lines. Tumour Biol 29, 195-203.

Longerich S, San Filippo J, Liu D, Sung P (2009) FANCI binds branched DNA and is monoubiquitinated by UBE2T-FANCL. J Biol Chem 284, 23182-6.

Machida YJ, Machida Y, Chen Y, Gurtan AM, Kupfer GM, D’Andrea AD, Dutta A (2006) UBE2T is the E2 in the Fanconi anemia pathway and undergoes negative autoregulation. Mol Cell 23, 589-96.

Zhang QH, Ye M, Wu XY, Ren SX, Zhao M, Zhao CJ, Fu G, Shen Y, Fan HY, Lu G, Zhong M, Xu XR, Han ZG, Zhang JW, Tao J, Huang QH, Zhou J, Hu GX, Gu J, Chen SJ, Chen Z (2000) Cloning and functional analysis of cDNAs with open reading frames for 300 previously undefined genes expressed in CD34+ hematopoietic stem/progenitor cells. Genome Res 10, 1546-60.