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

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.

How to control an intracellular proteolytic system: Coordinated regulatory switches in the mycobacterial Pup-proteasome system.

Intracellular proteolysis is critical for the proper functioning of all cells, owing to its involvement in a wide range of processes. Because of the destructive nature of protein degradation, intracellular proteolysis is restricted by control mechanisms at almost every step of the proteolytic process. Understanding the coordination of such mechanisms is a challenging task, especially in systems as complex as the eukaryotic ubiquitin-proteasome system (UPS). In comparison, the bacterial analog of the UPS, the Pup-proteasome system (PPS) is much simpler and, therefore, allows for insight into the control of a proteolytic system. This review integrates available information to present a coherent picture of what is known of PPS regulatory switches and describes how these switches act in concert to enforce regulation at the system level. Finally, open questions regarding PPS regulation are discussed, providing readers with a sense of what lies ahead in the field.

Proteasome accessory factor A (PafA) transferase activity makes sense in the light of its homology with glutamine synthetase.

The Pup-proteasome system (PPS) is a prokaryotic tagging and degradation system analogous in function to the ubiquitin-proteasome system (UPS). Like ubiquitin, Pup is conjugated to proteins, tagging them for proteasomal degradation. However, in the PPS, a single Pup-ligase, PafA, conjugates Pup to a wide variety of proteins. PafA couples ATP hydrolysis to formation of an isopeptide bond between Pup and a protein lysine via a mechanism similar to that used by glutamine synthetase (GS) to generate glutamine from ammonia and glutamate. GS can also transfer the glutamyl moiety from glutamine to a hydroxyl amine in an ATP-independent manner. Recently, the ability of PafA to transfer Pup from one protein to another was demonstrated. Here, we report that such PafA activity mechanistically resembles the transferase activity of GS. Both PafA and GS transferase activities are ATP-independent and proceed in two catalytic steps. In the first step catalyzed by PafA, an inorganic phosphate is used by the enzyme to depupylate a Pup donor, while forming an acyl phosphate Pup intermediate. The second step consists of Pup conjugation to the new protein, alongside the release of an inorganic phosphate. Detailed experimental analysis, combined with kinetic modeling of PafA transferase activity, allowed us to correctly predict the kinetics and magnitude of Pup transfer between two targets, and analyze the effects of their affinity to PafA on the efficiency of transfer. By deciphering the mechanism of the PafA transferase reaction in kinetic detail, this work provides in-depth mechanistic understanding of PafA, a key PPS enzyme.

An Extended Loop of the Pup Ligase, PafA, Mediates Interaction with Protein Targets.

Pupylation, the bacterial equivalent of ubiquitylation, involves the conjugation of a prokaryotic ubiquitin-like protein (Pup) to protein targets. In contrast to the ubiquitin system, where many ubiquitin ligases exist, a single bacterial ligase, PafA, catalyzes the conjugation of Pup to a wide array of protein targets. As mediators of target recognition by PafA have not been identified, it would appear that PafA alone determines pupylation target selection. Previous studies indicated that broad specificity and promiscuity are indeed inherent PafA characteristics that probably dictate which proteins are selected for degradation by the Pup-proteasome system. Nonetheless, despite the canonical role played by PafA in the Pup-proteasome system, the molecular mechanism that dictates target binding by PafA remains uncharacterized since the discovery of this enzyme about a decade ago. In this study, we report the identification of PafA residues involved in the binding of protein targets. Initially, docking analysis predicted the residues on PafA with high potential for target binding. Mutational and biochemical approaches subsequently confirmed these predictions and identified a series of additional residues located on an extended loop at the edge of the PafA active site. Mutating residues in this loop rendered PafA defective in the pupylation of a wide variety of protein targets but not in its catalytic mechanism, suggesting an important role for this extended loop in the binding of protein targets. As such, these findings pave the way toward an understanding of the molecular determinants that dictate the broad substrate specificity of PafA.

A kinetic model for the prevalence of mono- over poly-pupylation.

Bacteria belonging to the phyla Actinobacteria and Nitrospira possess proteasome cores homologous to the eukaryotic 20S proteasome particle. In these bacteria, the cytoplasmic signal for proteasomal degradation is a small protein termed Pup (prokaryotic ubiquitin-like protein). PafA, the only known Pup ligase, conjugates Pup to lysine side chains of target proteins. In contrast to the eukaryotic ubiquitin-proteasome system, where poly-ubiquitin chains are the principal tags for proteasomal degradation, mono-Pup moieties are almost exclusively observed in vivo and are sufficient as degradation tags. Although Pup presents lysines, raising the possibility of poly-Pup chain assembly, these do not predominate. At present, the factors promoting the distinct predominance of mono- over poly-pupylation remain poorly understood. To address this issue, we conducted a detailed biochemical analysis characterizing the pupylation of model proteins in vitro. We found that Pup can indeed serve as a pupylation target for PafA either in its free form or when already conjugated to proteins, thus allowing for the formation of poly-Pup chains. However, our results indicate that pupylation of an already pupylated protein is unlikely to occur due to low affinity of PafA for such species. This alone prevents predominance of poly- over mono-pupylation in vitro. This effect is likely to be magnified in vivo by the combination of PafA kinetics with the high abundance of non-pupylated proteins. Overall, this work provides a kinetic explanation for the prevalence of mono- rather than poly-pupylation in vivo, and sheds light on PafA substrate specificity.