Publications

• Bacterial ubiquitin-like modifier Pup is deamidated and conjugated to substrates by distinct but homologous enzymes.

  Striebel F., Imkamp F., Sutter M., Steiner M., Mamedov A., Weber-Ban E.

  In analogy to ubiquitin in eukaryotes, the bacterial protein Pup is attached to lysine residues of substrate proteins, thereby targeting them for proteasomal degradation. It has been proposed that, before its attachment, Pup is modified by deamidation of its C-terminal glutamine to glutamate. Here ... >> More

  In analogy to ubiquitin in eukaryotes, the bacterial protein Pup is attached to lysine residues of substrate proteins, thereby targeting them for proteasomal degradation. It has been proposed that, before its attachment, Pup is modified by deamidation of its C-terminal glutamine to glutamate. Here we have identified Dop (locus tag Rv2112) as the specific deamidase of Pup in Mycobacterium tuberculosis. Deamidation requires ATP as a cofactor but not its hydrolysis. Furthermore, we provide experimental evidence that PafA (locus tag Rv2097) ligates deamidated Pup to the proteasomal substrate proteins FabD and PanB. This formation of an isopeptide bond requires hydrolysis of ATP to ADP, suggesting that deamidated Pup is activated for conjugation via phosphorylation of its C-terminal glutamate. By combining these enzymes, we have reconstituted the complete bacterial ubiquitin-like modification pathway in vitro, consisting of deamidation and ligation steps catalyzed by Pup deamidase (Dop) and Pup ligase (PafA). << Less

  Nat. Struct. Mol. Biol. 16:647-651(2009) [PubMed] [EuropePMC]

• Pupylation as a signal for proteasomal degradation in bacteria.

  Striebel F., Imkamp F., Ozcelik D., Weber-Ban E.

  Posttranslational modifications in the form of covalently attached proteins like ubiquitin (Ub), were long considered an exclusive feature of eukaryotic organisms. The discovery of pupylation, the modification of lysine residues with a prokaryotic, ubiquitin-like protein (Pup), demonstrated that c ... >> More

  Posttranslational modifications in the form of covalently attached proteins like ubiquitin (Ub), were long considered an exclusive feature of eukaryotic organisms. The discovery of pupylation, the modification of lysine residues with a prokaryotic, ubiquitin-like protein (Pup), demonstrated that certain bacteria use a tagging pathway functionally related to ubiquitination in order to target proteins for proteasomal degradation. However, functional analogies do not translate into structural or mechanistic relatedness. Bacterial Pup, unlike eukaryotic Ub, does not adopt a β-grasp fold, but is intrinsically disordered. Furthermore, isopeptide bond formation in the pupylation process is carried out by enzymes evolutionary descendent from glutamine synthetases. While in eukaryotes, the proteasome is the main energy-dependent protein degradation machine, bacterial proteasomes exist in addition to other architecturally related degradation complexes, and their specific role along with the role of pupylation is still poorly understood. In Mycobacterium tuberculosis (Mtb), the Pup-proteasome system contributes to pathogenicity by supporting the bacterium's persistence within host macrophages. Here, we describe the mechanism and structural framework of pupylation and the targeting of pupylated proteins to the proteasome complex. Particular attention is given to the comparison of the bacterial Pup-proteasome system and the eukaryotic ubiquitin-proteasome system. Furthermore, the involvement of pupylation and proteasomal degradation in Mtb pathogenesis is discussed together with efforts to establish the Pup-proteasome system as a drug target. This article is part of a Special Issue entitled: Ubiquitin-Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf. << Less

  Biochim Biophys Acta 1843:103-113(2014) [PubMed] [EuropePMC]

• Deletion of dop in Mycobacterium smegmatis abolishes pupylation of protein substrates in vivo.

  Imkamp F., Rosenberger T., Striebel F., Keller P.M., Amstutz B., Sander P., Weber-Ban E.

  Proteasome-bearing bacteria make use of a ubiquitin-like modification pathway to target proteins for proteasomal turnover. In a process termed pupylation, proteasomal substrates are covalently modified with the small protein Pup that serves as a degradation signal. Pup is attached to substrate pro ... >> More

  Proteasome-bearing bacteria make use of a ubiquitin-like modification pathway to target proteins for proteasomal turnover. In a process termed pupylation, proteasomal substrates are covalently modified with the small protein Pup that serves as a degradation signal. Pup is attached to substrate proteins by action of PafA. Prior to its attachment, Pup needs to undergo deamidation at its C-terminal residue, converting glutamine to glutamate. This step is catalysed in vitro by Dop. In order to characterize Dop activity in vivo, we generated a dop deletion mutant in Mycobacterium smegmatis. In the Deltadop strain, pupylation is severely impaired and the steady-state levels of two known proteasomal substrates are drastically increased. Pupylation can be re-established by complementing the mutant with either DopWt or a Pup variant carrying a glutamate at its ultimate C-terminal position (PupGGE). Our data show that Pup is deamidated by Dop in vivo and that likely Dop alone is responsible for this activity. Furthermore, we demonstrate that a putative N-terminal ATP-binding motif is crucial for catalysis, as a single point mutation (E10A) in this motif abolishes Dop activity both in vivo and in vitro. << Less

  Mol. Microbiol. 75:744-754(2010) [PubMed] [EuropePMC]

• 'Depupylation' of prokaryotic ubiquitin-like protein from mycobacterial proteasome substrates.

  Burns K.E., Cerda-Maira F.A., Wang T., Li H., Bishai W.R., Darwin K.H.

  Ubiquitin (Ub) provides the recognition and specificity required to deliver proteins to the eukaryotic proteasome for destruction. Prokaryotic ubiquitin-like protein (Pup) is functionally analogous to Ub in Mycobacterium tuberculosis (Mtb), as it dooms proteins to the Mtb proteasome. Studies sugge ... >> More

  Ubiquitin (Ub) provides the recognition and specificity required to deliver proteins to the eukaryotic proteasome for destruction. Prokaryotic ubiquitin-like protein (Pup) is functionally analogous to Ub in Mycobacterium tuberculosis (Mtb), as it dooms proteins to the Mtb proteasome. Studies suggest that Pup and Ub do not share similar mechanisms of activation and conjugation to target proteins. Dop (deamidase of Pup; Mtb Rv2112c/MT2172) deamidates the C-terminal glutamine of Pup to glutamate, preparing it for ligation to target proteins by proteasome accessory factor A (PafA). While studies have shed light on the conjugation of Pup to proteins, it was not known if Pup could be removed from substrates in a manner analogous to the deconjugation of Ub from eukaryotic proteins. Here, we show that Mycobacteria have a "depupylase" activity provided by Dop. The discovery of a depupylase strengthens the parallels between the Pup- and Ub-tagging systems of prokaryotes and eukaryotes, respectively. << Less

  Mol. Cell 39:821-827(2010) [PubMed] [EuropePMC]