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 K6 linkages account for 11% of all yeast ubiquitin-ubiquitin linkages. The relative abundance of the other linkages were K11 (28%), K27 (9%), K29 (3%), K33 (4%), K48 (29%) and K63 (16%) (Xu et al., 2009). A well characterised heterodimeric RING E3 ligase complex consisting of the breast cancer-susceptibility proteins BRCA1 and BARD1 was shown to mediate K6-linked polyubiquitylation. However, the physiological roles of this chain type are currently unclear. Individuals who carry mutations in the BRCA1 gene are predisposed to early-onset breast and ovarian cancer. BRCA1/BARD1 are localized at sites of DNA damage, through binding of their adaptor protein RAP80 to K6- and K63-linked ubiquitin chains. Hence, both chain types may be involved in DNA Repair (Wu-Baer et al., 2003). The importance of the K6 residue per se has been investigated and it has been found that expression of a K6W mutant version of ubiquitin inhibits ubiquitin-dependent substrate proteolysis in mammalian cells. Furthermore Lys6-modified ubiquitin in cell-free assays significantly and specifically inhibited ATP-dependent proteolysis (Shang et al., 2005). It is speculated that the mechanism underlying the above phenomena may be related to the attenuated avidity of the interaction between K6-modified ubiquitin conjugates and the 26S proteasome. Furthermore it has been found that of the seven Lys residues in ubiquitin, Lys6 is the most readily modifiable with sulfo-NHS-biotin and other chemical reagents. Thus one can speculate that in vivo modifications of ubiquitin Lys6 are not only plausible but may interfere with proteasomal degradation and result in the accumulation of ubiquitin conjugates. Perhaps explaining why ubiquitin conjugates are accumulated in response to oxidative stress and upon aging (Shang et al., 2005).
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