Di-ubiquitin (K27-linked) [untagged]


Catalogue Number
60-0103-010
Product Size
10 µg
Price £
£165
Accession Number
P62987
Residues Expressed
n/a
Certificate of Analysis Size
10 µg
Species
human
Source
synthetic/chemical ligation
Quantity
10 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
~17.1 kDa
Stability
12 months at -70°C; aliquot as required
Protein Sequence
Accession number: P62987. For full protein sequence information download the Certificate of Analysis pdf.
QA; Protein Identification
The linkage type (K27) was confirmed by tandem mass spectrometry.
QA Activity

Di-ubiquitin cleavage assay: The capacity of the di-ubiquitin substrate to be cleaved was tested using a promiscuous - with respect to ubiquitin linkage specificity - deubiquitylase (GST-USP2). Incubation of the di-ubiquitin for 1 hour at 37°C was compared either in the absence (Lane 2) or presence (Lane 3) of GST-USP2. The reaction products were compared alongside two control samples containing either mono-ubiquitin (Lane 4) or GST-USP2 (Lane 5) only. Cleavage of the di-ubiquitin and generation of mono-ubiquitin was determined by running reactions on a 4-12% ­SDS-PAGE gel and staining with InstantBlue™ (Lane 1; molecular weight markers).


Background

Ubiquitin (Ub) is a highly conserved 76 amino-acid protein found throughout eukaryotic cells. A vast number of cellular processes, including targeted protein degradation, cell cycle progression, DNA repair, protein trafficking, inflammatory response, virus budding, and receptor endocytosis, are regulated by Ub-mediated signalling; where the target protein is tagged by single or multi-monomeric Ub (monomeric Ub attached to multiple sites on the substrate) or a polymeric chain of Ubs (Fushman et al., 2010). This post-translational modification is tightly controlled by an enzymatic cascade involving several enzymes (E1, E2, and E3) and occurs through either an isopeptide bond between the C-terminal Glycyl residue of Ub and the epsilon amino group of a Lysyl residue on a target protein or through a peptide bond between the C-terminal Glycyl residue of Ub and the N-terminal amine on a further Ub. In the former (isopeptide bond-linked) case the substrate protein may either be ubiquitin itself - thus leading to the generation of poly-ubiquitin chains - or another target protein (Fushman et al., 2010). Thus, ubiquitin can be attached to a substrate either as a monomer or as a poly-ubiquitin chain. Further - depending on their linkage type (M1, K6, K11, K27, K29, K33, K48 and K63 linked) - the Ub chains can take different structural forms. Chains containing all eight possible Ub linkages have been found in living cells and different ubiquitin chain types may encode different biological signals, allowing this single protein to mediate many diverse functions (Komander 2009; Weeks et al., 2009; Walczak et al., 2012). The functionality of Ub chains is most commonly associated with their attachment to substrate proteins but there is also evidence that they may also play a role in cellular signalling as free chains (Braten et al., 2012). A mass spectrometry-based study found that K27 linkages account for 9% of all yeast ubiquitin-ubiquitin linkages. The relative abundance of the other linkages were K6 (11%), K11 (28%), K29 (3%), K33 (4%), K48 (29%) and K63 (16%) (Xu et al., 2009). The role of the K27 linkage remains elusive, possibly in part due to the close proximity of K29 and K33 linkages making MS analysis a challenge (owing to the nature of the tryptic digest fragments). No cellular role has been associated with K27-linked polymers to date (Komander 2009).


References

Braten O, Shabek N, Kravtsova-Ivantsiv Y, Ciechanover A (2012) Generation of free ubiquitin chains is upregulated in stress, and facilitated by the HECT domain ubiquitin ligases UFD4 and HUL5. Biochem J 444, 611-617.

Fushman D, Walker O (2010) Exploring the linkage dependence of polyubiquitin conformations using molecular modeling. Journal of Molecular Biology 395, 803-814.

Komander D (2009) The emerging complexity of protein ubiquitination. Biochem Soc Trans 37, 937-953.

Walczak H, Iwai K, Dikic I (2012) Generation and physiological roles of linear ubiquitin chains. BMC Biol 10, 23.

Weeks SD, Grasty KC, Hernandez-Cuebas L, Loll PJ (2009) Crystal structures of Lys-63-linked tri- and di-ubiquitin reveal a highly extended chain architecture. Proteins 77, 753-759.

Xu P, Duong DM, Seyfried NT, Cheng D, Xie Y, et al., (2009) Quantitative proteomics reveals the function of unconventional ubiquitin chains in proteasomal degradation. Cell 137, 133-145.