IDOL [6His-tagged]


Catalogue Number
63-0044-025
Product Size
25 µg
Price £
£250
Accession Number
NP_037394.2
Residues Expressed
1-445
Certificate of Analysis Size
25 µg
Species
human
Source
E.coli
Quantity
25 µg
Storage
-70°C
Concentration
1mg/ml
Formulation
50 mM HEPES pH 7.5, 150 mM sodium chloride, 2 mM dithiothreitol, 10% glycerol
Molecular Weight
~54kDa
Stability
12 months at -70°C; aliquot as required
Protein Sequence
Accession number: NP_037394.2. 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-IDOL 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-UBE2D2 (UbcH5b) (several E2s were tested, data generated with this E2 is provided by way of example) and ubiquitin. Incubation of His-IDOL for 30 minutes at 30oC in the presence of ubiquitin, His-UBE1, His-UBE2D2 and ATP (Lane 1) was compared alongside two control reactions with either ATP (Lane 2) or His-IDOL (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-IDOL.


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). Inducible Degrader of LDLR (IDOL) is a member of the E3 protein ligase family and cloning of the human gene was first described by Olsson et al. (1999). IDOL is a RING finger domain ubiquitin E3 ligase that is up-regulated by the sterol-activated transcription factors LXR alpha and LXR beta. IDOL activity leads to ubiquitylation and degradation of the low density lipoprotein (LDL) receptor (LDLR). LDLR is essential for the uptake of LDL cholesterol and the regulation of plasma lipoprotein levels and lipid homeostasis (Zelcer et al., 2009; Zhang et al., 2011). An inherited loss-of-function mutation in the LDLR gene in humans or poor diet can elevate plasma LDL levels, reduce LDL clearance and accelerate atherosclerosis and the risk of cardiovascular disease (Tolleshaug et al., 1983; Brown and Goldstein 1986).


References

Brown MS, Goldstein JL. (1986) A receptor-mediated pathway for cholesterol homeostasis. Science 232, 34-47.

Olsson PA, Korhonen L, Mercer E A, Lindholm D. (1999) MIR is a novel ERM-like protein that interacts with myosin regulatory light chain and inhibits neurite outgrowth. J Biol Chem 274, 36288-36292.

Tolleshaug H, Hobgood KK, Brown MS, Goldstein JL. (1983) The LDL receptor locus in familial hypercholesterolemia: multiple mutations disrupt transport and processing of a membrane receptor. Cell 32, 941-51.

Zelcer N, Hong C, Boyadjian R, Tontonoz P. (2009) LXR regulates cholesterol uptake through Idol-dependent ubiquitination of the LDL receptor. Science 325, 100-4.

Zhang L, Fairall L, Goult BT, Calkin AC, Hong C, Millard CJ, Tontonoz P, Schwabe JW. (2011) The IDOL-UBE2D complex mediates sterol-dependent degradation of the LDL receptor. Genes Dev 25, 1262-74.