Chemical strategies to understand the language of ubiquitin signaling


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Ubiquitin (Ub) is a small 76 amino acid long protein that is highly conserved in all eukaryotes studied to date. In humans, more than 600 ligases are involved in the reversible modification of specific lysine side-chain amines in substrate proteins by conjugation with the C-terminal carboxylate of Ub. Initially monoubiquitylated proteins can undergo repetitive ubiquitylation starting from one of seven lysine residues or the α-amine in the first Ub to generate a variety of polyUb chains with different topologies and functions. The most well known role for protein ubiquitylation is in targeting substrates for proteolytic destruction by 26S proteasomes. However, a growing body of evidence indicates that both mono- and polyubiquitylation play proteasome-independent roles in modulating the structure, function, and localization of protein substrates. Understanding the complexity of Ub-mediated functions in our cells is a major challenge for modern biology. In addition to well-established in vivo genetic methods, biochemical and biophysical investigations of ubiquitylated proteins in vitro can shed light on the direct mechanistic roles for Ub in different contexts. Such studies have traditionally been limited by the ability to obtain sufficient quantities of homogenously ubiquitylated proteins with precisely defined linkages. This review focuses on recent advances in both synthetic and recombinant protein-based methods that have yielded access to homogenously site-specifically ubiquitylated proteins. Mechanistic studies of the roles for protein ubiquitylation and of the enzymes involved in protein deubiquitylation that are enabled by these chemical tools are highlighted. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 144–155, 2014.