UBE2E3 (UbcH9) [untagged]


Catalogue Number
62-0022-100
Product Size
100 µg
Price £
£325
Accession Number
NP_006348
Residues Expressed
1-207
Alternate Product Size
20 µg
Certificate of Analysis Size
100 µg
Species
Human
Source
E. coli expression
Quantity
100 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_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 UBE2E3 was validated by loading E1 UBE1 activated ubiquitin onto the active cysteine of the UBE2E3 E2 enzyme via a transthiolation reaction. Incubation of the UBE1 and 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/UBE2E3 thioester bond to the reducing agent DTT was confirmed.

 

Background

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 α 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.