Dataset Information

Amino acid residues involved in inactivation of the Escherichia coli multidrug resistance repressor MarR by salicylate, 2,4-dinitrophenol, and plumbagin.

ABSTRACT: MarR is the dedicated autorepressor of the marRAB operon found in seven genera of the Enterobacteraceae. The MarA transcriptional regulator directly activates numerous genes involved in multidrug resistance and other environmental responses. MarR is inactivated by certain phenolic ligands, such as salicylate, by an unknown mechanism. Our recent work has shown that several amino acid residues of Escherichia coli MarR affecting ligand binding are located between the dimerization and DNA-binding domains. To further characterize the ligand-binding region of MarR, we have now examined 7 point mutants generated by random mutagenesis and 11 site-directed alanine replacement mutants for inactivation by three ligands: salicylate, 2,4-dinitrophenol, and plumbagin. Inactivation of MarR was quantitated in intact cells by loss of MarR-mediated repression of a chromosomal mar-lacZ transcriptional fusion. The results showed that most of the residues important for ligand effectiveness lay in the ?1 and ?2 helices of MarR, between the putative DNA-binding domain and the dimerization domain of MarR, reinforcing our earlier findings. Moreover, the three ligands had different, but overlapping, sets of residues impacting their effects on MarR.

SUBMITTER: McMurry LM

PROVIDER: S-EPMC3947609 | biostudies-literature | 2013 Dec

REPOSITORIES: biostudies-literature

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Amino acid residues involved in inactivation of the Escherichia coli multidrug resistance repressor MarR by salicylate, 2,4-dinitrophenol, and plumbagin.

McMurry Laura M LM Levy Stuart B SB

FEMS microbiology letters 20131021 1

MarR is the dedicated autorepressor of the marRAB operon found in seven genera of the Enterobacteraceae. The MarA transcriptional regulator directly activates numerous genes involved in multidrug resistance and other environmental responses. MarR is inactivated by certain phenolic ligands, such as salicylate, by an unknown mechanism. Our recent work has shown that several amino acid residues of Escherichia coli MarR affecting ligand binding are located between the dimerization and DNA-binding do ...[more]

PMID: 24111786

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Project description:1. The effect of dinitrophenol on the metabolism of glucose labelled with (14)C and tritium by epididymal fat-pad segments from fed rats was studied. Dinitrophenol at concentrations of 0.1-0.3mm: (a) had little effect on glucose utilization; (b) depressed synthesis of fatty acids and greatly increased that of lactate; (c) increased the T/(14)C ratio in fatty acids synthesized from [U-(14)C,3-T]glucose and decreased that in fatty acids synthesized from [U-(14)C,4-T]glucose; (d) abolished randomization of (14)C from [6-(14)C]glucose in lactate. 2. Dinitrophenol stimulated oxidation of pyruvate and greatly inhibited the oxidation of lactate. It inhibited lipogenesis from pyruvate and lactate. 3. From the isotope data it was calculated that: (a) dinitrophenol stimulates oxidation via the tricarboxylic acid cycle three- to six-fold; (b) dinitrophenol depresses markedly the operation of the pentose cycle; (c) in the presence of dinitrophenol, NADPH formed in the pentose cycle provides all the hydrogen equivalents for fatty acid reduction, whereas, in its absence, NADPH provides 50-70% of the hydrogen equivalents; (d) in the presence of dinitrophenol, there is an excess of ATP produced in the cytoplasm, which flows into the mitochondria. A reverse flow operates in the absence of dinitrophenol. 4. A balance of formation and utilization of reduced nicotinamide nucleotides in the cytoplasm was established. With dinitrophenol there is some excess of NADH. There are indications that this excess may be transferred into mitochondria in the form of malate. 5. Our results are interpreted to indicate the absence from adipose tissue of the alpha-glycerophosphate shuttle for transferring reducing equivalents from the cytoplasm to mitochondria. 6. The effects of dinitrophenol are accounted for in terms of decreased ATP concentrations in the cells, leading to marked decrease in pyruvate carboxylation in the mitochondria and depression of fatty acid synthesis in the cytoplasm.

Project description:Background2,4-Dinitrophenol (2,4-DNP) is an effective but highly dangerous fat burner, not licensed for human consumption. Death cases reported for 2,4-DNP overdose, particularly among young adults, have raised concerns about the ineffective regulatory control, lack of education and risks associated with impurity, and the unknown concentration of 2,4-DNP purchased on the Internet.MethodsUsing a sequential mixed method design and based on a hypothetical scenario as if 2,4-DNP was a licensed pharmaceutical drug, first we conducted a qualitative study to explore what product attributes people consider when buying a weight-loss aid. Focus group interviews with six females and three males (mean age = 21.6 ± 1.8 years) were audiorecorded, transcribed verbatim, and subjected to thematic analysis. Sixteen attributes were identified for the Best-Worst Scale (BWS) in the quantitative survey with 106 participants (64% female, mean age = 27.1 ± 11.9 years), focusing on 2,4-DNP. Demographics, weight satisfaction, and risk for eating disorder data were collected.ResultsIn contrast to experienced users such as bodybuilders, our study participants approached 2,4-DNP cautiously. Attributes of 2,4-DNP as a hypothetical weight-loss drug comprised a range of desirable and avoidable features. Of the 16 selected attributes, BWS suggested that long-term side effects were the most and branding was the least important attribute. Effectiveness and short-term side effects were also essential. Those in the >25 year group showed least concerns for legality. Neutral BWS scores for cost, treatment, degree of lifestyle changes required, and specificity required for the hypothetical weight-loss drug to be effective were likely caused by disagreement about their importance among the participants, not indifference.ConclusionWith advances in research, 2,4-DNP as a pharmaceutical drug in the future for treating neurodegenerative diseases and potentially for weight loss is not inconceivable. Caution is warranted for interpreting the BWS scores. Owing to the difference in what data represent at individual vs. population levels, with pooled data, the method correctly identifies attributes by which most people are satisfied but misrepresents attributes that are individually very important but not universally agreed. Whilst this may be an advantage in marketing applications, it limits the utility of BWS as a research tool.

Dataset Information

Amino acid residues involved in inactivation of the Escherichia coli multidrug resistance repressor MarR by salicylate, 2,4-dinitrophenol, and plumbagin.

Publications

Amino acid residues involved in inactivation of the Escherichia coli multidrug resistance repressor MarR by salicylate, 2,4-dinitrophenol, and plumbagin.

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