TRIAD1 [6His-tagged]


Catalogue Number
63-0029-025
Product Size
25 µg
Price £
£175
Accession Number
NP_006312
Residues Expressed
1-493
Certificate of Analysis Size
25 µg
Species
Human
Source
Sf21 insect cell-baculovirus expression
Quantity
25 μg
Storage
-70°C
Concentration
0.5 mg/ml
Formulation
50 mM HEPES pH 7.5, 150 mM sodium chloride, 2 mM dithiothreitol, 10% glycerol
Molecular Weight
~61.2 kDa
Stability
12 months at -70°C; aliquot as required
Protein Sequence
Accession number: NP_006312. For full protein sequence information download the Certificate of Analysis pdf.
QA; Protein Identification
Confirmed by mass spectrometry.
QA Activity

E3 ligase assay: The ubiquitin conjugating activity of His-TRIAD1 was validated through its ability to catalyse the generation of polyubiquitin chains in the presence of the E1 activating enzyme His-UBE1, the E2 conjugating enzyme His-UBE2D1 (UbcH5a) (several E2s were tested, data generated with this E2 is provided by way of example) and ubiquitin. Incubation of His-TRIAD1 for 120 minutes at 37°C in the presence of ubiquitin, His-UBE1, His-UBE2D1 and ATP (Lane 1) was compared alongside two control reactions with either ATP (Lane 2) or His-TRIAD1 (Lane 3) excluded from the reaction. Ubiquitin conjugates were identified by Western blotting using an anti-ubiquitin conjugate antibody and these were observed only in the presence of both ATP and His-TRIAD1.


Background

The enzymes of the ubiquitylation pathway play a pivotal role in a number of cellular processes including the regulated and targeted proteasome-dependent 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). Triad Domain-Containing Protein 1 (TRIAD1) is a member of the E3 protein ligase family and cloning of the human gene was first described by van der Reijden et al. (1999). TRIAD1 contains a TRIAD motif containing two RING domains which flank a conserved cysteine-rich (C6HC) domain designated DRIL (double RING finger-linked domain) (Marteijn et al., 2005). TRIAD is thought to be involved in protein translation, interacting with UbcH7 (UBE2L3) to polyubiquitylate eIF4E2 targeting it for proteasomal degradation (Tan et al., 2003). More recently these proteins have been referred to as Ring in-between Ring E3 ligases (RBRs) that function like RING-HECT hybrids regulating processes such as translation and immune signalling (Wenzel et al., 2011).


References

Marteijn JA, van Emst L, Erpelinck-Verschueren CA, Nikoloski G, Menke A, de Witte T, Lowenberg B, Jansen JH, van der Reijden BA (2005) The E3 ubiquitin-protein ligase Triad1 inhibits clonogenic growth of primary myeloid progenitor cells. Blood 106, 4114-23.

Tan NG, Ardley HC, Scott GB, Rose SA, Markham AF, Robinson PA (2003) Human homologue of ariadne promotes the ubiquitylation of translation initiation factor 4E homologous protein, 4EHP. FEBS Lett 554, 501-4.

van der Reijden BA, Erpelinck-Verschueren CA, Lowenberg B, Jansen JH (1999) TRIADs: a new class of proteins with a novel cysteine-rich signature. Protein Sci 8, 1557-61.

Wenzel DM, Lissounov A, Brzovic PS, Klevit RE (2011) UBCH7 reactivity profile reveals parkin and HHARI to be RING/HECT hybrids. Nature 474, 105-8.