Inter- and intramolecular regulation of protein depupylation in Mycobacterium smegmatis.

Whereas intracellular proteolysis is essential for proper cellular function, it is a destructive process, which must be tightly regulated. In some bacteria, a Pup-proteasome system tags target proteins for degradation by a bacterial proteasome. Pup, a small modifier protein, is attached to target proteins by PafA, the sole Pup ligase, in a process termed pupylation. In mycobacteria, including Mycobacterium smegmatis and Mycobacterium tuberculosis, Pup undergoes a deamidation step by the enzyme Dop prior to its PafA-mediated attachment to a target. The catalytic mechanism of Pup deamidation is also used by Dop to perform depupylation, namely the removal of Pup from already tagged proteins. Hence, Dop appears to play contradictory roles: On the one hand, deamidation of Pup promotes pupylation, while on the other hand, depupylation reduces tagged protein levels. To avoid futile pupylation-depupylation cycles, Dop activity must be regulated. An intramolecular regulatory mechanism directs Dop to catalyze deamidation more effectively than depupylation. A complementary intermolecular mechanism results in Dop depletion under conditions where protein pupylation and degradation are favorable. In this work, we studied these regulatory mechanisms and identified a flexible loop in Dop, previously termed the Dop-loop, that acts as an intramolecular regulatory element that allosterically controls substrate preference. To investigate regulation at the intermolecular level, we used the CRISPR interference system to knock down the expression of M. smegmatis ATP-dependent intracellular proteases and found that the ClpCP protease is responsible for Dop depletion under starvation conditions. These findings clarify previous observations and introduce a new level for the regulation of Dop activity. DATABASE: Structural data are available in the PDB database under the accession numbers 4BJR and 4B0S.

Multiple layers of regulation determine the cellular levels of the Pup ligase PafA in Mycobacterium smegmatis.

Despite being a destructive process, regulated protein degradation is fundamental for proper cell function. While regulated proteolysis in eukaryotes largely involves the ubiquitin-proteasome system (UPS), most bacterial species rely on multiple ATP-dependent proteases, such as the Clp proteases. Mycobacteria and related actinobacterial species also possess a degradation system analogous in its function to the UPS. In this system, a prokaryotic ubiquitin-like protein (Pup) is conjugated to proteins, thereby marking them for proteasomal degradation. A single ligase, PafA, is responsible for Pup conjugation to many protein targets, thus playing a central role in the Pup-proteasome system (PPS). In Mycobacterium smegmatis, a model mycobacterial organism where the PPS is essential under starvation conditions, cellular PafA levels change in response to nutrient availability. Indeed, increased PafA levels are observed upon nutrient limitation. We found that a multi-layered network involving transcriptional, translational and post-translational regulation determines cellular PafA levels. Induced expression is observed at stationary phase, whereas PafA degradation by the proteasome and ClpCP occurs in exponentially growing cells, as opposed to starved cells. In both growth stages, translation attenuation maintains low PafA expression levels. Altogether, these mechanisms establish the dynamics in PafA levels during bacterial growth.

The transcription of pafA, encoding the prokaryotic ubiquitin-like protein ligase, is regulated by PafBC.

AIM: Mycobacterium tuberculosis possesses an intracellular tagging and degradation system, which has emerged as a target for development of anti-tuberculosis agents. In this system, PafA is the ligase that marks proteins for degradation by their covalent modification with a protein modifier. Here, we studied pafA transcriptional regulation, which remained elusive despite its importance for M. tuberculosis virulence. MATERIALS & METHODS: Working with Mycobacterium smegmatis, a mycobacterial model organism, we examined the involvement of the global regulators PafB and PafC in pafA regulation. RESULTS: PafBC activated pafA transcription following DNA damage, resulting in efficient cellular recovery. CONCLUSION: The results unraveled the involvement of PafBC in pafA transcription, and revealed the importance of proper PafA regulation in mycobacterial physiology.

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.

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.

Bacterial proteasome and PafA, the pup ligase, interact to form a modular protein tagging and degradation machine.

Proteasome-containing bacteria possess a tagging system that directs proteins to proteasomal degradation by conjugating them to a prokaryotic ubiquitin-like protein (Pup). A single ligating enzyme, PafA, is responsible for Pup conjugation to lysine side chains of protein substrates. As Pup is recognized by the regulatory subunit of the proteasome, Pup functions as a degradation tag. Pup presents overlapping regions for binding of the proteasome and PafA. It was, therefore, unclear whether Pup binding by the proteasome regulatory subunit, Mpa, and by PafA are mutually exclusive events. The work presented here provides evidence for the simultaneous interaction of Pup with both Mpa and PafA. Surprisingly, we found that PafA and Mpa can form a complex both in vitro and in vivo. Our results thus suggest that PafA and the proteasome can function as a modular machine for the tagging and degradation of cytoplasmic proteins.

Allosteric transitions direct protein tagging by PafA, the prokaryotic ubiquitin-like protein (Pup) ligase.

Protein degradation via prokaryotic ubiquitin-like protein (Pup) tagging is conserved in bacteria belonging to the phyla Actinobacteria and Nitrospira. The physiological role of this novel proteolytic pathway is not yet clear, although in Mycobacterium tuberculosis, the world's most threatening bacterial pathogen, Pup tagging is important for virulence. PafA, the Pup ligase, couples ATP hydrolysis with Pup conjugation to lysine side chains of protein substrates. PafA is the sole Pup ligase in M. tuberculosis and apparently, in other bacteria. Thus, whereas PafA is a key player in the Pup tagging (i.e. pupylation) system, control of its activity and interactions with target protein substrates remain poorly understood. In this study, we examined the mechanism of protein pupylation by PafA in Mycobacterium smegmatis, a model mycobacterial organism. We report that PafA is an allosteric enzyme that binds its target substrates cooperatively and find that PafA allostery is controlled by the binding of target protein substrates, yet is unaffected by Pup binding. Analysis of PafA pupylation using engineered substrates differing in the number of pupylation sites points to PafA acting as a dimer. These findings suggest that protein pupylation can be regulated at the level of PafA allostery.