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 multimonomeric Ub (monomeric Ub attached to multiple sites on the substrate) or a polymeric chain of Ubs (Fushman and Walker, 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).
Ubiquitin also acts as a second messenger in the proteasomal degradation pathway. In a dual mechanism, the kinase PTEN Induced putative Kinase1 (PINK1) phosphorylates Ser65 on Ubiquitin and Ser65 in the Ubl domain of the E3 ligase Parkin. Phosphorylation of Parkin at Ser65 results in the opening of the Parkin structure to allow the binding of Ser65 phosphorylated Ubiquitin thus activating the E3 ligase (Kane et al., 2014; Kazlauskaite et al., 2014; Koyano et al., 2014). Phospho-ubiquitin may play other roles in regulating Parkin but more generally the identification of phospho-ubiquitin as a second messenger in signalling pathways could reveal the existence of further ubiquitin phosphatases and lead to the discovery of additional substrates and signalling functions (Sauve and Gehring, 2014).
Biotin-Ahx-ubiquitin (synthetic) (Cat# 60-0201-050) is a non-phosphorylated synthetically made ubiquitin which may be used as an experimental control alongside any of the Biotin-Ahxphosphorylated ubiquitin reagents Biotin-Ahx-ubiquitin (pThr7) (Cat# 60-0203-050), Biotin-Ahx-ubiquitin (pThr12) (Cat# 60-0204-050), BiotinAhx-ubiquitin (pSer57) (Cat# 60-0205-050), Biotin-Ahx-ubiquitin (pTyr59) (Cat# 60-0206-050), Biotin-Ahx-ubiquitin (pSer65) (Cat# 60-0207-050) or any other modified ubiquitin with an N-terminal ‘Biotin-Ahx' tag.
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Kazlauskaite A, Kondapalli C, Gourlay R, Campbell DG, Ritorto MS, Hofmann K, et al. (2014) Parkin is activated by PINK1-dependent phosphorylation of ubiquitin at Ser65. Biochem J, 460, 127-139.
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Koyano F, Okatsu K, Kosako H, Tamura Y, Go E, Kimura M, et al.(2014) Ubiquitin is phosphorylated by PINK1 to activate parkin. Nature, 510, 162-166.
Sauve V and Gehring K (2014) Phosphorylated ubiquitin: a new shade of PINK1 in Parkin activation. Cell Res, 24, 1025-6.
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.