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Trans-aconitate methyltransferase regulator (TamR) is a member of the ligand-responsive multiple antibiotic resistance regulator (MarR) family of transcription factors. In Streptomyces coelicolor, TamR regulates transcription of tamR (encoding TamR), tam (encoding trans-aconitate methyltransferase) and sacA (encoding aconitase); up-regulation of these genes promotes metabolic flux through the citric acid cycle. DNA binding by TamR is attenuated and transcriptional derepression is achieved on binding of ligands such as citrate and trans-aconitate to TamR. In the present study, we show that three additional genes are regulated by S. coelicolor TamR. Genes encoding malate synthase (aceB1; SCO6243), malate dehydrogenase (mdh; SCO4827) and isocitrate dehydrogenase (idh; SCO7000) are up-regulated in vivo when citrate and trans-aconitate accumulate, and TamR binds the corresponding gene promoters in vitro, a DNA binding that is attenuated by cognate ligands. Mutations to the TamR binding site attenuate DNA binding in vitro and result in constitutive promoter activity in vivo. The predicted TamR binding sites are highly conserved in the promoters of these genes in Streptomyces species that encode divergent tam-tamR gene pairs, suggesting evolutionary conservation. Like aconitase and trans-aconitate methyltransferase, malate dehydrogenase, isocitrate dehydrogenase and malate synthase are closely related to the citric acid cycle, either catalysing individual reaction steps or, in the case of malate synthase, participating in the glyoxylate cycle to produce malate that enters the citric acid cycle to replenish the intermediate pool. Taken together, our data suggest that TamR plays an important and conserved role in promoting metabolic flux through the citric acid cycle.

Ras GTPases undergo post-translational modifications that govern their subcellular trafficking and localization. In particular, palmitoylation of the Golgi tags N-Ras and H-Ras for exocytotic transport and residency at the PM (plasma membrane). Following depalmitoylation, PM-Ras redistributes to all subcellular membranes causing an accumulation of palmitate-free Ras at endomembranes, including the Golgi and endoplasmic reticulum. Palmitoylation is unanimously regarded as a critical modification at the crossroads of Ras activity and trafficking control, but its precise relevance to native wild-type Ras function in growth factor signalling is unknown. We show in the present study by use of palmitoylation-deficient N-Ras mutants and via the analysis of palmitate content of agonist-activated GTP-loaded N-Ras that only palmitoylated N-Ras becomes activated by agonists. In line with an essential role of palmitoylation in Ras activation, dominant-negative RasS17N loses its blocking potency if rendered devoid of palmitoylation. Live-cell Ras-GTP imaging shows that N-Ras activation proceeds only at the PM, consistent with activated N-Ras-GTP being palmitoylated. Finally, palmitoylation-deficient N-Ras does not sustain EGF (epidermal growth factor) or serum-elicited mitogenic signalling, confirming that palmitoylation is essential for signal transduction by N-Ras. These findings document that N-Ras activation proceeds at the PM and suggest that depalmitoylation, by removing Ras from the PM, may contribute to the shutdown of Ras signalling.

The atherogenic cytokine IL-6 (interleukin-6) induces pro-inflammatory gene expression in VECs (vascular endothelial cells) by activating the JAK (Janus kinase)/STAT3 (signal transducer and activator of transcription 3) signalling pathway, which is normally down-regulated by the STAT3-dependent induction of the E3 ubiquitin ligase component SOCS3 (suppressor of cytokine signalling 3). Novel treatments based on the regulation of SOCS3 protein levels could therefore have value in the treatment of diseases with an inflammatory component, such as atherosclerosis. To this end we carried out a screen of 1031 existing medicinal compounds to identify inducers of SOCS3 gene expression and identified the flavanoids naringenin and flavone as effective inducers of SOCS3 protein, mRNA and promoter activity. This was in contrast with the action of traditional JAK/STAT3 inhibitors and the polyphenol resveratrol, which effectively suppress SOCS3 gene expression. Both naringenin and flavone also effectively suppressed IL-6-stimulated phosphorylation of STAT3 (Tyr⁷⁰⁵) which led to suppression of IL-6-induction of the atherogenic STAT3 target gene MCP1 (monocyte chemotactic protein-1), suggesting that their ability to induce SOCS3 gene expression is STAT3-independent. Supporting this idea was the observation that the general kinase inhibitor compound C inhibits flavone- and cAMP-dependent, but not JAK-dependent, SOCS3 induction in VECs. Indeed, the ability of flavanoids to induce SOCS3 expression requires activation of the ERK (extracellular-signal-regulated kinase)-dependent transcription factor SP3, and not STAT3. In the present paper we therefore describe novel molecular actions of flavanoids, which control SOCS3 gene induction and suppression of STAT3 signalling in VECs. These mechanisms could potentially be exploited to develop novel anti-atherogenic therapies.

GPCRs (G-protein-coupled receptors) exist in a spontaneous equilibrium between active and inactive conformations that are stabilized by agonists and inverse agonists respectively. Because ligand binding of agonists and inverse agonists often occurs in a competitive manner, one can assume an overlap between both binding sites. Only a few studies report mutations in GPCRs that convert receptor blockers into agonists by unknown mechanisms. Taking advantage of a genetically modified yeast strain, we screened libraries of mutant M(3)Rs {M(3) mAChRs [muscarinic ACh (acetylcholine) receptors)]} and identified 13 mutants which could be activated by atropine (EC50 0.3-10 microM), an inverse agonist on wild-type M(3)R. Many of the mutations sensitizing M(3)R to atropine activation were located at the junction of intracellular loop 3 and helix 6, a region known to be involved in G-protein coupling. In addition to atropine, the pharmacological switch was found for other M(3)R blockers such as scopolamine, pirenzepine and oxybutynine. However, atropine functions as an agonist on the mutant M(3)R only when expressed in yeast, but not in mammalian COS-7 cells, although high-affinity ligand binding was comparable in both expression systems. Interestingly, we found that atropine still blocks carbachol-induced activation of the M(3)R mutants in the yeast expression system by binding at the high-affinity-binding site (Ki approximately 10 nM). Our results indicate that blocker-to-agonist converting mutations enable atropine to function as both agonist and antagonist by interaction with two functionally distinct binding sites.