“Depupylation” of Prokaryotic Ubiquitin-like Protein from Mycobacterial Proteasome Substrates

Abstract

Summary 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 carboxyl-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.

RhodococcusProkaryotic Ubiquitin-Like Protein (Pup) Is Degraded by Deaminase of Pup (Dop)

“…1C), as reported by other researchers. 11,12) We confirmed that the band contained Pup Mt by performing Western blot and LC-MS/MS analyses (data not shown). The results obtained for Dop Re were similar to those obtained for Dop Mt , but free Pup was not detected when Pup Re conjugated proteins were used as substrate (Fig.…”

“…These peptidase activities were completely abolished when depupylase-inactive mutant Dop ReE10A was tested (data not shown). 11,28) Pup has a disordered structure and is not highly conserved among actinomycetes (Fig. 2B), 30,31) as indicated by the various Pup cleavage patterns generated by Dop.…”

“…10) In this Pup-dependent proteolytic system, Dop functions not only as a deaminase of Pup but also as a depupylase, removing Pup from pupylated substrates by cleaving the isopeptide bond between Pup and the target proteins. 11,12) Given that the proteasome and proteasomal ATPase are not essential in Rhodococcus, it is unclear how the half-life of pupylated proteins is regulated in cells lacking genes that encode these proteins. Moreover, no all pupylated proteins lead to degradation.…”

Genetic and Proteomic Analyses of Pupylation in Streptomyces coelicolor

“…This observation could reflect the fact that the genome of S. coelicolor is nearly twice the size of those of M. tuberculosis and M. smegmatis. An inherent challenge in the identification of all pupylated proteins in S. coelicolor or any other actinobacterium is that pupylation is a reversible phenomenon (28). This consideration led us to carry out the proteomic analyses on lysates from mycelia in different phases of growth.…”

Profiling the Proteome of Mycobacterium tuberculosis during Dormancy and Reactivation

“…Proteasome accessory factor A (PafA), the Pup ligase, subsequently ligates the newly-formed side chain carboxylate to a lysine residue of the target protein (48). Pupylated proteins are guided into the proteasome through the binding of Pup to the proteasomal ATPase, which unfolds proteins prior to delivery into the proteasome core (49). Dop also functions as a depupylase to remove Pup from substrate proteins prior to proteasomal degradation (50).…”