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Proteasomal control of cytokinin synthesis protects Mycobacterium tuberculosis against nitric oxide.

ABSTRACT: One of several roles of the Mycobacterium tuberculosis proteasome is to defend against host-produced nitric oxide (NO), a free radical that can damage numerous biological macromolecules. Mutations that inactivate proteasomal degradation in Mycobacterium tuberculosis result in bacteria that are hypersensitive to NO and attenuated for growth in vivo, but it was not known why. To elucidate the link between proteasome function, NO resistance, and pathogenesis, we screened for suppressors of NO hypersensitivity in a mycobacterial proteasome ATPase mutant and identified mutations in Rv1205. We determined that Rv1205 encodes a pupylated proteasome substrate. Rv1205 is a homolog of the plant enzyme LONELY GUY, which catalyzes the production of hormones called cytokinins. Remarkably, we report that an obligate human pathogen secretes several cytokinins. Finally, we determined that the Rv1205-dependent accumulation of cytokinin breakdown products is likely responsible for the sensitization of Mycobacterium tuberculosis proteasome-associated mutants to NO.

SUBMITTER: Samanovic MI

PROVIDER: S-EPMC4369403 | biostudies-literature | 2015 Mar

REPOSITORIES: biostudies-literature

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Proteasomal control of cytokinin synthesis protects Mycobacterium tuberculosis against nitric oxide.

Samanovic Marie I MI Tu Shengjiang S Novák Ondřej O Iyer Lakshminarayan M LM McAllister Fiona E FE Aravind L L Gygi Steven P SP Hubbard Stevan R SR Strnad Miroslav M Darwin K Heran KH

Molecular cell 20150226 6

One of several roles of the Mycobacterium tuberculosis proteasome is to defend against host-produced nitric oxide (NO), a free radical that can damage numerous biological macromolecules. Mutations that inactivate proteasomal degradation in Mycobacterium tuberculosis result in bacteria that are hypersensitive to NO and attenuated for growth in vivo, but it was not known why. To elucidate the link between proteasome function, NO resistance, and pathogenesis, we screened for suppressors of NO hyper ...[more]

PMID: 25728768

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Project description:Mycobacterium tuberculosis group I truncated hemoglobin trHbN catalyzes the oxidation of nitric oxide (NO) to nitrate with a second-order rate constant k approximately 745 microM(-1) s(-1) at 23 degrees C (nitric oxide dioxygenase reaction). It was proposed that this high efficiency is associated with the presence of hydrophobic tunnels inside trHbN structure that allow substrate diffusion to the distal heme pocket. In this work, we investigated the mechanisms of NO diffusion within trHbN tunnels in the context of the nitric oxide dioxygenase reaction using two independent approaches. Molecular dynamics simulations of trHbN were performed in the presence of explicit NO molecules. Successful NO diffusion from the bulk solvent to the distal heme pocket was observed in all simulations performed. The simulations revealed that NO interacts with trHbN at specific surface sites, composed of hydrophobic residues located at tunnel entrances. The entry and the internal diffusion of NO inside trHbN were performed using the Long, Short, and EH tunnels reported earlier. The Short tunnel was preferentially used by NO to reach the distal heme pocket. This preference is ascribed to its hydrophobic funnel-shape entrance, covering a large area extending far from the tunnel entrance. This funnel-shape entrance triggers the frequent formation of solvent-excluded cavities capable of hosting up to three NO molecules, thereby accelerating NO capture and entry. The importance of hydrophobicity of entrances for NO capture is highlighted by a comparison with a polar mutant for which residues at entrances were mutated with polar residues. A complete map of NO diffusion pathways inside trHbN matrix was calculated, and NO molecules were found to diffuse from Xe cavity to Xe cavity. This scheme was in perfect agreement with the three-dimensional free-energy distribution calculated using implicit ligand sampling. The trajectories showed that NO significantly alters the dynamics of the key amino acids of Phe(62)(E15), a residue proposed to act as a gate controlling ligand traffic inside the Long tunnel, and also of Ile(119)(H11), at the entrance of the Short tunnel. It is noteworthy that NO diffusion inside trHbN tunnels is much faster than that reported previously for myoglobin. The results presented in this work shed light on the diffusion mechanism of apolar gaseous substrates inside protein matrix.

Project description:The Mycobacterium tuberculosis H37Rv genome has been sequenced and annotated over 20 years ago, yet roughly half of the protein-coding genes still lack a predicted function. We characterized two genes of unknown function, rv3679 and rv3680, for which inconsistent findings regarding their importance for virulence in mice have been reported. We confirmed that a rv3679-80 deletion mutant (Δrv3679-80) was virulent in mice and discovered that Δrv3679-80 suffered from a glycerol-dependent recovery defect on agar plates following mouse infection. Glycerol also exacerbated killing of Δrv3679-80 by nitric oxide. Rv3679-Rv3680 have previously been shown to form a complex with ATPase activity and we demonstrate that the ability of M. tuberculosis to cope with elevated levels of glycerol and nitric oxide requires intact ATP-binding motifs in both Rv3679 and Rv3680. Inactivation of glycerol kinase or Rv2370c, a protein of unknown function, suppressed glycerol mediated toxicity in Δrv3679-80 Glycerol catabolism led to increased intracellular methylglyoxal pools and Δrv3679-80 was hypersusceptible to extracellular methylglyoxal suggesting that glycerol toxicity in Δrv3679-80 is caused by methylglyoxal. Rv3679 and Rv3680 interacted with Rv1509, and Rv3679 had numerous additional interactors including proteins of the type II fatty acid synthase (FASII) pathway and mycolic acid modifying enzymes linking Rv3679 to fatty acid or lipid synthesis. This work provides experimentally determined roles for Rv3679 and Rv3680 and stimulates future research on these and other proteins of unknown function.Importance A better understanding of the pathogenesis of tuberculosis requires a better understanding of gene function in M. tuberculosis This work provides the first functional insight into the Rv3679/Rv3680 ATPase complex. We demonstrate that M. tuberculosis requires this complex and specifically its ATPase activity to resist glycerol and nitric oxide toxicity. We provide evidence that the glycerol-derived metabolite methylglyoxal causes toxicity in the absence of Rv3679/Rv3680. We further show that glycerol-dependent toxicity is reversed when glycerol kinase (GlpK) is inactivated. Our work uncovered other genes of unknown function that interact with Rv3679 and/or Rv3680 genetically or physically, underscoring the importance of understanding uncharacterized genes.

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Proteasomal control of cytokinin synthesis protects Mycobacterium tuberculosis against nitric oxide.

Publications

Proteasomal control of cytokinin synthesis protects Mycobacterium tuberculosis against nitric oxide.

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