title: Molecular analysis of the prokaryotic ubiquitin-like protein (Pup) conjugation pathway in Mycobacterium tuberculosis.
abstract: Proteins 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.
authors.affiliation: New York University School of Medicine, Department of Microbiology, 550 First Avenue, New York, NY 10016, USA.
authors.forename: Francisca A
authors.initials: FA
authors.lastname: Cerda-Maira
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Pearce
authors.forename: Michele
authors.initials: M
authors.lastname: Fuortes
authors.forename: William R
authors.initials: WR
authors.lastname: Bishai
authors.forename: Stevan R
authors.initials: SR
authors.lastname: Hubbard
authors.forename: K Heran
authors.initials: KH
authors.lastname: Darwin
mesh_terms: D000581:Amidohydrolases / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D058491:Bacterial Load; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004195:Disease Models, Animal; D008168:Lung / Q000382:microbiology; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013154:Spleen / Q000382:microbiology; D014376:Tuberculosis / Q000382:microbiology / Q000473:pathology; D014452:Ubiquitins / Q000235:genetics / Q000378:metabolism*; D014774:Virulence; D037521:Virulence Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D050505:Mutant Proteins; C533288:Pup protein, Mycobacterium tuberculosis; D014452:Ubiquitins; D037521:Virulence Factors; D000581:Amidohydrolases; C556013:Dop protein, Mycobacterium tuberculosis