Dataset Information

Toxin ? Reduces the ATP and Modulates the Uridine Diphosphate-N-acetylglucosamine Pool.

ABSTRACT: Toxin ? expression triggers a reversible state of dormancy, diminishes the pool of purine nucleotides, promotes (p)ppGpp synthesis, phosphorylates a fraction of the peptidoglycan precursor uridine diphosphate-N-acetylglucosamine (UNAG), leading to unreactive UNAG-P, induces persistence in a reduced subpopulation, and sensitizes cells to different antibiotics. Here, we combined computational analyses with biochemical experiments to examine the mechanism of toxin ? action. Free ? toxin showed low affinity for UNAG. Toxin ? bound to UNAG hydrolyzed ATP·Mg2+, with the accumulation of ADP, Pi, and produced low levels of phosphorylated UNAG (UNAG-P). Toxin ?, which has a large ATP binding pocket, may temporally favor ATP binding in a position that is distant from UNAG, hindering UNAG phosphorylation upon ATP hydrolysis. The residues D67, E116, R158 and R171, involved in the interaction with metal, ATP, and UNAG, were essential for the toxic and ATPase activities of toxin ?; whereas the E100 and T128 residues were partially dispensable. The results indicate that ? bound to UNAG reduces the ATP concentration, which indirectly induces a reversible dormant state, and modulates the pool of UNAG.

SUBMITTER: Moreno-Del Alamo M

PROVIDER: S-EPMC6356619 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Publications

Toxin ζ Reduces the ATP and Modulates the Uridine Diphosphate-N-acetylglucosamine Pool.

Moreno-Del Álamo María M Tabone Mariangela M Muñoz-Martínez Juan J Valverde José R JR Alonso Juan C JC

Toxins 20190109 1

Toxin ζ expression triggers a reversible state of dormancy, diminishes the pool of purine nucleotides, promotes (p)ppGpp synthesis, phosphorylates a fraction of the peptidoglycan precursor uridine diphosphate-N-acetylglucosamine (UNAG), leading to unreactive UNAG-P, induces persistence in a reduced subpopulation, and sensitizes cells to different antibiotics. Here, we combined computational analyses with biochemical experiments to examine the mechanism of toxin ζ action. Free ζ toxin showed low ...[more]

PMID: 30634431

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Project description:BACKGROUND: Uridine diphosphate (UDP) is an extracellular nucleotide signaling molecule implicated in diverse biological processes via specific activation of pyrimidinergic receptor P2Y, G Protein-Coupled, 6 (P2Y6). There is very little knowledge about the function and mechanism of UDP in rheumatoid arthritis (RA). METHODS: This study used a quasi-targeted liquid chromatography-mass spectrometry (LC-MS) approach to investigate the unique expression of metabolites in RA synovial fluids (SF) (n = 10) with samples from osteoarthritis (OA) as controls (n = 10). RA fibroblast-like synoviocytes (FLSs) were collected from synovial tissues (n = 5) and cultured with UDP or MRS2578, a P2Y6 antagonist, and FLSs from OA were used as controls (n = 5). Rats with collagen-induced arthritis (CIA) were injected with UDP, MRS2578 or both (n = 9 for each group). P2Y6 expression was examined using real-time PCR, Western blotting and immunohistochemistry. Cell proliferation, apoptosis and migration of RA FLSs were measured using CCK-8 assay, real-time cell analysis, flow cytometry, wound healing assay and Transwell assay, respectively. The UDP levels in the culture medium, synovial fluid (n = 36) and peripheral blood (n = 36) of RA and CIA rats were measured using a Transcreener UDP Assay. Levels of proinflammatory cytokines were measured using a flow assay. Interleukin-6 (IL-6) levels were measured using ELISA and flow. RESULTS: LC-MS analysis detected significantly increased UDP levels in RA SF compared with OA SF, and the level was positively correlated with anticyclic citrullinated peptide (anti-CCP) and rheumatoid factor (RF)levels in RA. The increased UDP concentration was verified in the blood and synovial fluids of RA patients compared with samples from OA patients and healthy volunteers, respectively. UDP stimulated cell proliferation, migration and IL-6 secretion in RA FLSs and inhibited their apoptosis in culture, and MRS2578 inhibited these effects of UDP. UDP injection accelerated CIA and stimulated IL-6 production rather than other proinflammatory cytokines in the rat model, but simultaneous injection of MRS2578 suppressed these effects and alleviated CIA. P2Y6 expression was increased in RA and CIA synovial tissues. CONCLUSION: These results suggest that UDP is highly expressed in RA and stimulates RA pathogenesis by promoting P2Y6 activities to increase IL-6 production.

Dataset Information

Toxin ? Reduces the ATP and Modulates the Uridine Diphosphate-N-acetylglucosamine Pool.

Publications

Toxin ζ Reduces the ATP and Modulates the Uridine Diphosphate-N-acetylglucosamine Pool.

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