Dataset Information

Reconstitution of the Mycobacterium tuberculosis pupylation pathway in Escherichia coli.

ABSTRACT: Prokaryotic ubiquitin-like protein (Pup) is a post-translational modifier that attaches to more than 50 proteins in Mycobacteria. Proteasome accessory factor A (PafA) is responsible for Pup conjugation to substrates, but the manner in which proteins are selected for pupylation is unknown. To address this issue, we reconstituted the pupylation of model Mycobacterium proteasome substrates in Escherichia coli, which does not encode Pup or PafA. Surprisingly, Pup and PafA were sufficient to pupylate at least 51 E. coli proteins in addition to the mycobacterial proteins. These data suggest that pupylation signals are intrinsic to targeted proteins and might not require Mycobacterium-specific cofactors for substrate recognition by PafA in vivo.

SUBMITTER: Cerda-Maira FA

PROVIDER: S-EPMC3147258 | biostudies-literature | 2011 Jul

REPOSITORIES: biostudies-literature

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Reconstitution of the Mycobacterium tuberculosis pupylation pathway in Escherichia coli.

Cerda-Maira Francisca A FA McAllister Fiona F Bode Nadine J NJ Burns Kristin E KE Gygi Steven P SP Darwin K Heran KH

EMBO reports 20110708 8

Prokaryotic ubiquitin-like protein (Pup) is a post-translational modifier that attaches to more than 50 proteins in Mycobacteria. Proteasome accessory factor A (PafA) is responsible for Pup conjugation to substrates, but the manner in which proteins are selected for pupylation is unknown. To address this issue, we reconstituted the pupylation of model Mycobacterium proteasome substrates in Escherichia coli, which does not encode Pup or PafA. Surprisingly, Pup and PafA were sufficient to pupylate ...[more]

PMID: 21738222

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Project description:Mycobacterium tuberculosis nicotinamidase-pyrazinamidase (PZAse) is a metalloenzyme that catalyzes conversion of nicotinamide-pyrazinamide to nicotinic acid-pyrazinoic acid. This study investigated whether a metallochaperone is required for optimal PZAse activity. M. tuberculosis and Escherichia coli PZAses (PZAse-MT and PZAse-EC, respectively) were inactivated by metal depletion (giving PZAse-MT-Apo and PZAse-EC-Apo). Reactivation with the E. coli metallochaperone ZnuA or Rv2059 (the M. tuberculosis analog) was measured. This was repeated following proteolytic and thermal treatment of ZnuA and Rv2059. The CDC1551 M. tuberculosis reference strain had the Rv2059 coding gene knocked out, and PZA susceptibility and the pyrazinoic acid (POA) efflux rate were measured. ZnuA (200 μM) achieved 65% PZAse-EC-Apo reactivation. Rv2059 (1 μM) and ZnuA (1 μM) achieved 69% and 34.3% PZAse-MT-Apo reactivation, respectively. Proteolytic treatment of ZnuA and Rv2059 and application of three (but not one) thermal shocks to ZnuA significantly reduced the capacity to reactivate PZAse-MT-Apo. An M. tuberculosis Rv2059 knockout strain was Wayne positive and susceptible to PZA and did not have a significantly different POA efflux rate than the reference strain, although a trend toward a lower efflux rate was observed after knockout. The metallochaperone Rv2059 restored the activity of metal-depleted PZAse in vitro Although Rv2059 is important in vitro, it seems to have a smaller effect on PZA susceptibility in vivo. It may be important to mechanisms of action and resistance to pyrazinamide in M. tuberculosis Further studies are needed for confirmation.IMPORTANCE Tuberculosis is an infectious disease caused by the bacterium Mycobacterium tuberculosis and remains one of the major causes of disease and death worldwide. Pyrazinamide is a key drug used in the treatment of tuberculosis, yet its mechanism of action is not fully understood, and testing strains of M. tuberculosis for pyrazinamide resistance is not easy with the tools that are presently available. The significance of the present research is that a metallochaperone-like protein may be crucial to pyrazinamide's mechanisms of action and of resistance. This may support the development of improved tools to detect pyrazinamide resistance, which would have significant implications for the clinical management of patients with tuberculosis: drug regimens that are appropriately tailored to the resistance profile of a patient's individual strain lead to better clinical outcomes, reduced onward transmission of infection, and reduction of the development of resistant strains that are more challenging and expensive to treat.

Dataset Information

Reconstitution of the Mycobacterium tuberculosis pupylation pathway in Escherichia coli.

Publications

Reconstitution of the Mycobacterium tuberculosis pupylation pathway in Escherichia coli.

Similar Datasets

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