Chemical ubiquitination for decrypting a cellular code

Abstract

The modification of proteins with ubiquitin (Ub) is an important regulator of eukaryotic biology and deleterious perturbation of this process is widely linked to the onset of various diseases. The regulatory capacity of the Ub signal is high and, in part, arises from the capability of Ub to be enzymatically polymerised to form polyubiquitin (polyUb) chains of eight different linkage types. These distinct polyUb topologies can then be site-specifically conjugated to substrate proteins to elicit a number of cellular outcomes. Therefore, to further elucidate the biological significance of substrate ubiquitination, methodologies that allow the production of defined polyUb species, and substrate proteins that are site-specifically modified with them, are essential to progress our understanding. Many chemically inspired methods have recently emerged which fulfil many of the criteria necessary for achieving deeper insight into Ub biology. With a view to providing immediate impact in traditional biology research labs, the aim of this review is to provide an overview of the techniques that are available for preparing Ub conjugates and polyUb chains with focus on approaches that use recombinant protein building blocks. These approaches either produce a native isopeptide, or analogue thereof, that can be hydrolysable or non-hydrolysable by deubiquitinases. The most significant biological insights that have already been garnered using such approaches will also be summarized.

De novo macrocyclic peptides that specifically modulate Lys48-linked ubiquitin chains

“…3e, f). However, USP2 in the presence of the cyclic peptides Ub2i, Ub4ix, and Ub4i could only remove the distal Ub of K48 Ub 4 40 , shortening the chain to the trimer K48 Ub 3 , the peptides show strong inhibition of further disassembly of the chain (Fig. 3e, f).…”

From atoms to physiology: what it takes to really understand inflammasomes

“…While modified peptides can easily be synthesized to elicit conventional antibodies, immunization to generate nanobodies will require intact proteins exhibiting desired post‐translational modifications, relying on the in vitro modification with purified enzymes. Where the respective enzymes are not available or known, novel methods to generate artificially modified proteins using non‐natural amino acids and semi‐synthetic approaches to achieve site‐specific ubiquitination or phosphorylation will prove useful (Stanley & Virdee, ; Mukai et al . ).…”

Avidity-Based Method for the Efficient Generation of Monoubiquitinated Recombinant Proteins

“…Additionally, chemical (nonenzymatic) methods that can produce isopeptide-linked monoubiquitinated proteins are technically challenging and not applicable to most proteins. 6 There is, therefore, a need for a technique that can facilitate the high-yield production of monoubiquitinated proteins. Here we present an efficient approach that fills this gap.…”

“…This is because enzymatic reactions performed on recombinant substrates often generate a composite mixture containing reaction components and mono-, multimono-, and polyubiquitinated products, and selective purification of monoubiquitinated species from this soup is difficult. Additionally, chemical (nonenzymatic) methods that can produce isopeptide-linked monoubiquitinated proteins are technically challenging and not applicable to most proteins . There is, therefore, a need for a technique that can facilitate the high-yield production of monoubiquitinated proteins.…”