Together the protein engineering approaches employed
Together, the protein engineering approaches employed by Teyra et al., (2019) demonstrate the effectiveness of affinity maturation for improving the affinity of UbVs, but this comes at a cost in terms of specificity. Indeed, the most effective UbV was the dimeric construct generated from UbVs isolated from the initial library. Surprisingly, in contrast to most reported UbVs, none of the USP15 UbVs bind in the known ubiquitin-binding sites. It is unclear if in USP15 the UbVs have selected cryptic ubiquitin or ubiquitin-like Valsartan or whether the UbVs are more comparable to nanobodies in that they have selected a surface-exposed epitope that is unrelated to ubiquitin binding.
Acknowledgments We apologize to colleagues working in this field whose work was not cited due to space constraints. Our work is supported by grants from the Marsden Fund (NZ) and from the Health Research Council of New Zealand.
Introduction Proteins are vital for the structure and function of the cells, and the regulation of protein synthesis is a prime aspect of cellular metabolism. About 30% of mammalian proteins are short lived, have very short half-life of less than 10min and are rapidly degraded after translation (Schubert et al., 2000). Such a high level of protein degradation requires a dedicated system to regulate the selective unwanted protein degradation. Ubiquitin-proteasome system (UPS) has emerged as a key supervisor of protein function and stability. UPS has many vital roles in eukaryotic cellular processes including cell cycle progression, stress response, signal transduction, DNA repair, control of transcription factor activity and membrane trafficking (Coux et al., 1996, Hershko and Ciechanover, 1998, Ciechanover et al., 2000a, Ciechanover, 2006, Welchman et al., 2005). Ubiquitin plays an important role to degrade proteins through proteasome targeting as well as by direct sorting to the lysosome. Ubiquitin is a small eukaryotic polypeptide which marks unwanted or damaged proteins for degradation, and the proteasome, is a large molecule breaks down protein into smaller peptides, to be used in other anabolic processes (D'Arcy et al., 2015). More than 80% of proteins are degraded by UPS and that is why it has emerged as an important player in the regulation of various cellular processes (Rock et al., 1994). UPS plays a pivotal role in the pathogenesis of many human diseases like cancer and neurodegenerative disorders (Ciechanover et al., 2000b). The process of ubiquitination is a multi-step process ultimately leading to the covalent modification of a protein substrate with small molecule ubiquitin. There are three types of ubiquitination: 1) mono-ubiquitination in which single ubiquitin is attached to target 2) multi ubiquitination or poly-mono-ubiquitination where several single ubiquitin are attached to target proteins 3) poly ubiquitination where substrate is attached with poly-ubiquitin chains (D'Arcy et al., 2015, Jentsch and Schlenker, 1995, Di Fiore et al., 2003, Lander et al., 2012). Ubiquitin has ≅76 conserved amino acid protein that covalently attached through a peptide bond between the carboxyl glycine residues at 76 position of ubiquitin to the amino groups of lysine residues in target proteins. The process of ubiquitination depends on the consecutive activity of three distinct enzymes, ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2) and ubiquitin-ligase or E3 ubiquitin ligase (E3) (fig. 1). In the first step, ubiquitin is activated by the E1 in the presence of ATP, forming a thio-ester bond between the carboxyl-terminal glycine residue of ubiquitin and the active site cysteine of the E1 enzyme. Once activated, ubiquitin is transferred from E1 to a cysteine residue of E2 ubiquitin carrier proteins. Substrate specificity is mediated by E3 ligases, which bind target substrates and co-ordinate the covalent attachment of ubiquitin. Two distinct families of E3 ligases exist, the HECT domain family that receives ubiquitin from the E2 ligase forming an ubiquitin-E3 intermediate, and the RING finger family of E3 ligases that form a molecular bridge between the E2 ligase and target proteins (D'Arcy et al., 2015, Ross et al., 2015, Voges et al., 1999, Pickart and Eddins, 2004). Once ubiquitin-E2, E3 and substrate complex is formed, ubiquitin binds to the substrate which is mediated by E3 ligase. In the subsequent process E2 and E3 are removed from complex and substrate protein will be either degraded or alternatively substrated could be free from ubiquitin through deubiquitinases and remain active (fig. 1).