Ubiquitination via Chemical Ligation between Artificial Amino Acids

Abstract

Modification of proteins by the covalent attachment of ubiquitin (Ub) plays a fundamental role in the control of many biological processes including cell cycle regulation, transcription, DNA repair, and apoptosis. Substrate proteins are either mono-ubiquitinated or poly-ubiquitinated, i. e. several Ub monomers are attached to form poly-Ub chains. In these chains several Ub moieties are linked to each other via isopeptide bonds between a specific lysine residue of one Ub and the C-terminal glycine of the next Ub. Ub contains seven lysine residues and each of these lysines can be used for poly-Ub chain formation. Importantly, the actual lysine residue of Ub used for Ub-Ub conjugation seems to determine the biological function of the respective poly-Ub chain.The aim of the present work was to develop a method to synthesize all naturally occurring Ub dimers and to mono-ubiquitinate substrate proteins in vitro. This will provide the basis to elucidate different functions of differently-linked poly-Ub chains and of mono-ubiquitination.Cross-linking of Ub to other proteins (either a second Ub or a substrate protein) was achieved using the Cu(I)-catalyzed Huisgen cycloaddition, the so-called click reaction. The two orthogonal functional groups needed for click chemistry, an azide and an alkyne, had to be incorporated into the proteins via artificial amino acids. The azide function was introduced at the C-terminus of one Ub via the methionine analog azidohomoalanine (Aha) using selective pressure incorporation. The alkyne function was introduced via a pyrrolysine analog, the propargyl-protected lysine derivative Plk using amber suppression. It replaced the respective lysine residues naturally used for conjugation. Subsequent click reaction between the two modified proteins resulted in a hydrolytically stable triazole linkage. With this, the synthesis of all seven naturally occurring Ub dimers was possible, as well as the site-specific mono-ubiquitination of the two substrate proteins PCNA and DNA polymerase β.