Chemical strategies to understand the language of ubiquitin signaling

Abstract

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 ε-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.

Chemical and Genetic Approaches to Study Histone Modifications

“…On the other hand, monoubiquitylation is associated with multiple cellular signaling roles. Histones are modified by both mono-and polyubiquitin and our understanding of the roles for their ubiquitylation has grown significantly in the last decade [73]. Although all four core histones and the linker histone H1 are modified by Ub, historically monoubiquitylation at Lys120 in H2B (uH2B) and Lys119 in H2A (uH2A) have received the most attention [74,75].…”

Chemical Synthesis of Ubiquitin Chains

“…Moreover, identifying the appropriate enzyme combinations and applying the enzymatic machinery for ubiquitination of protein targets is highly challenging. In this regard, chemical synthesis has turned into a reliable tool to access such precious biomaterials where solid phase peptide synthesis (SPPS), peptide ligation, and protein semi‐synthesis are being utilized for the non‐enzymatic preparation of interesting Ub conjugates (Spasser and Brik, ; Strieter and Korasick, ; Hemantha and Brik, ; Abeywardana and Pratt, ; Weller et al., ). An obvious drawback of these methods is in their limited utility for medium‐sized protein substrates (200 to 300 residues).…”

Selenocysteine as a Latent Bioorthogonal Electrophilic Probe for Deubiquitylating Enzymes

“…15 This led us to consider replacing Gly76 in Ub with SeCys as a latent precursor to Dha. We envisioned using SeCys as a handle for site-specific substrate ubiquitylation, 16 followed by its chemoselective elimination to generate Dha (Scheme 1). …”