Pup-Click-A New Chemoenzymatic Method for the Generation of Singly Pupylated Targets.

Abstract

Conjugation of the prokaryotic ubiquitin-like protein (Pup) to cellular proteins tags these proteins for degradation by a proteasome in actinobacteria. To study the Pup-proteasome system in in vitro biochemical assays, Pup-tagged (i.e., pupylated) proteins are often used. However, the purification of a homogeneous preparation of pupylated proteins often suffers from poor yields and limitations in terms of selecting the target protein and its site of pupylation. Here, we report on the development of a biochemical methodology we term Pup-Click for the generation of pupylated protein mimics in vitro. Pup-Click relies on a natural pupylation reaction combined with the use of a synthetic peptide and genetic code expansion via the use of unnatural amino acids and Click chemistry. In principle, this approach allows for conjugation of Pup to any selected target at potentially any desired position. Importantly, pupylated protein mimics generated by Pup-Click are recognized and processed by enzymes of the Pup-proteasome system. As such, Pup-Click can serve as a powerful tool for studying this protein degradation pathway.