Carpe diubiquitin.

Eukaryotic cells are made more diverse and complex by an intricate ensemble of post-translational modifications to their proteins. Of these, ubiquitination is perhaps the most fateful. Ubiquitin (Ub) is a small, robust, highly conserved protein (Figure 1). The covalent attachment of ubiquitin marks cellular proteins for degradation by the proteasome, and can elicit other consequences as well. Defects in ubiquitin demarcation correlate with multiple diseases, including cancers, inflammatory diseases, and a variety of neurodegenerative disorders. Ubiquitination is forged through an isopeptide bond—a nonstandard amide linkage between the e-nitrogen of a lysine residue on a target “acceptor” protein and the C-terminal carboxyl group of ubiquitin “donor”. A lysine residue on the appended ubiquitin can be decorated subsequently with another ubiquitin, and so on. Ubiquitin has seven lysine residues (K6, K11, K27, K29, K33, K48, and K63; Figure 1). Each possible isopeptide bond exists in living cells, and particular linkages stimulate disparate responses. The production of proteins containing non-canonical linkages, such as isopeptide bonds, is an ongoing challenge in chemical biology. Recently, three independent groups have announced the construction of diubiquitin, including the total synthesis of all possible diubiquitin chains. This accomplishment is a milestone in chemical biology, enabling the first explorations of the structure and function of diubiquition regioisomers. The isopeptide bond is not accessible with the standard techniques of recombinant DNA. Isopeptide bond-forming enzymes can, however, provide access. In pioneering work, the Pickart group developed a chemoenzymatic route to diand polyubiquitin (Table 1). This work led to the discovery of K48-linked chains containing at least four ubiquitins as a signal for proteasomal degradation. Methods of modern protein chemistry are expanding the horizons of isopeptide bond formation. The initial products were precise conjugates with a single ubiquitin. In 2007, the Muir group used a photolabile auxiliary group to effect the semisynthesis of a histone with a ubiquitin pendant at a specific site, a modification that can have marked effects on transcription. In addition to being a harbinger, the Muir approach is noteworthy in being traceless—no residual atoms or groups remain in the synthetic target. Other chemical means to decorate targets with a single ubiquitin moiety have availed a pyrrolysine analogue that enables native chemical ligation, disulfide bond formation, or an isopeptide-like bond with an oxime. More recent work has expanded to the synthesis of diubiquitin. Using thioether formation as a mechanism for concatenating synthetic peptides, the Przybylski group completed a total synthesis of K63-linked diubiquitin. In their product, thioethers replace several native peptide bonds. The new work by the groups of Liu and Liu, Chin and Komander, and Brik is most exciting. Their distinct, complementary approaches have led to the traceless total synthesis of designated diubiquitin chains in quantities useful for structure–function analyses. Figure 1. Three-dimensional structure of ubiquitin, which contains 76 amino acid residues, with its seven lysine residues indicated explicitly (black; blue=NH2 groups).