Molecular analysis of the prokaryotic ubiquitin-like protein (Pup) conjugation pathway in Mycobacterium tuberculosis

Abstract

SummaryProteins targeted for degradation by the Mycobacterium proteasome are post-translationally tagged with prokaryotic ubiquitin-like protein (Pup), an intrinsically disordered protein of 64 residues. In a process termed 'pupylation', Pup is synthesized with a terminal glutamine, which is deamidated to glutamate by Dop (deamidase of Pup) prior to attachment to substrate lysines by proteasome accessory factor A (PafA). Importantly, PafA was previously shown to be essential to cause lethal infections by Mycobacterium tuberculosis (Mtb) in mice. In this study we show that Dop, like PafA, is required for the full virulence of Mtb. Additionally, we show that Dop is not only involved in the deamidation of Pup, but also needed to maintain wild-type steady state levels of pupylated proteins in Mtb. Finally, using structural models and site-directed mutagenesis our data suggest that Dop and PafA are members of the glutamine synthetase fold family of proteins.

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

“…The conserved residues are important to Dop activity, and are believed to be involved in the coordination of Mg 2þ , ATP, and the C-terminal amino acid residue of Pup, glutamate. 28) The depupylase activity of Dop Re was abolished by the addition of EDTA (Fig. 3A).…”

“…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.…”

Posttranslational Modification of Cellular Proteins by a Ubiquitin-like Protein in Bacteria

“…The mechanism of conjugation of TtuB to target proteins, which utilizes the E1-like TtuC protein, also differs from that of pupylation. In the pupylation reaction, the C-terminal Gln-64 of Pup is deamidated to Glu by the glutamate synthetase-like protein Dop (40 -42), and deamidated Pup is conjugated to Lys residues on target proteins by another glutamate synthetase-like protein, PafA (40,42). The side chain of C-terminal Glu of Pup is coupled to substrates (43).…”

The arginylation branch of the N-end rule pathway positively regulates cellular autophagic flux and clearance of proteotoxic proteins

“…1,2 The N-degrons are the first defined degradation signals in eukaryotes and have later been identified even in bacteria, which lack Ub. 3,4 These findings suggest that N-degron-dependent proteolysis may be an evolutionary precursor of nature's selective degradation machinery through the "chambered proteases" in the cell. 5 The N-end rule pathway in mammals has 2 branches, defined by the nature of N-degrons: the acetylation branch (Ac/N-end rule) and the arginylation branch (Arg/N-end rule).…”