Lithium Enhances the GABAergic Synaptic Activities on the Hypothalamic Preoptic Area (hPOA) Neurons.

Lithium (Li+) salt is widely used as a therapeutic agent for treating neurological and psychiatric disorders. Despite its therapeutic effects on neurological and psychiatric disorders, it can also disturb the neuroendocrine axis in patients under lithium therapy. The hypothalamic area contains GABAergic and glutamatergic neurons and their receptors, which regulate various hypothalamic functions such as the release of neurohormones, control circadian activities. At the neuronal level, several neurotransmitter systems are modulated by lithium exposure. However, the effect of Li+ on hypothalamic neuron excitability and the precise action mechanism involved in such an effect have not been fully understood yet. Therefore, Li+ action on hypothalamic neurons was investigated using a whole-cell patch-clamp technique. In hypothalamic neurons, Li+ increased the GABAergic synaptic activities via action potential independent presynaptic mechanisms. Next, concentration-dependent replacement of Na+ by Li+ in artificial cerebrospinal fluid increased frequencies of GABAergic miniature inhibitory postsynaptic currents without altering their amplitudes. Li+ perfusion induced inward currents in the majority of hypothalamic neurons independent of amino-acids receptor activation. These results suggests that Li+ treatment can directly affect the hypothalamic region of the brain and regulate the release of various neurohormones involved in synchronizing the neuroendocrine axis.

First evidence for the presence of amino acid sensing mechanisms in the fish gastrointestinal tract.

This study aimed to characterize amino acid sensing systems in the gastrointestinal tract (GIT) of the carnivorous fish model species rainbow trout. We observed that the trout GIT expresses mRNAs encoding some amino acid receptors described in mammals [calcium-sensing receptor (CaSR), G protein-coupled receptor family C group 6 member A (GPRC6A), and taste receptors type 1 members 1 and 2 (T1r1, T1r2)], while others [taste receptor type 1 member 3 (T1r3) and metabotropic glutamate receptors 1 and 4 (mGlur1, mGlur4)] could not be found. Then, we characterized the response of such receptors, as well as that of intracellular signaling mechanisms, to the intragastric administration of L-leucine, L-valine, L-proline or L-glutamate. Results demonstrated that casr, gprc6a, tas1r1 and tas1r2 mRNAs are modulated by amino acids in the stomach and proximal intestine, with important differences with respect to mammals. Likewise, gut amino acid receptors triggered signaling pathways likely mediated, at least partly, by phospholipase C ß3 and ß4. Finally, the luminal presence of amino acids led to important changes in ghrelin, cholecystokinin, peptide YY and proglucagon mRNAs and/or protein levels. Present results offer the first set of evidence in favor of the existence of amino acid sensing mechanisms within the fish GIT.

Inhibition of decidual IGF-1 signaling in response to hypoxia and leucine deprivation is mediated by mTOR and AAR pathways and increased IGFBP-1 phosphorylation.

Decidual mechanistic target of rapamycin (mTOR) is inhibited, amino acid response (AAR) and protein kinase CK2 are activated, and IGF (insulin-like growth factor) binding protein (IGFBP)-1 is hyperphosphorylated in human intrauterine growth restriction (IUGR). Using decidualized human immortalized endometrial stromal cells (HIESC), we hypothesized that hypoxia and leucine deprivation causing inhibition of decidual IGF-1 signaling is mediated by mTOR, AAR, CK2 and IGFBP-1 phosphorylation. Mass spectrometry demonstrated that hypoxia (1% O2) or rapamycin increased IGFBP-1 phosphorylation singly at Ser101/119/169 (confirmed using immunoblotting) and dually at pSer169 + 174. Hypoxia resulted in mTOR inhibition, AAR and CK2 activation, and decreased IGF-1 bioactivity, with no additional changes with rapamycin + hypoxia. Rapamycin and/or hypoxia promoted colocalization of IGFBP-1 and CK2 (dual-immunofluorescence and proximity ligation assay). Leucine deprivation showed similar outcomes. Changes in IGFBP-1 phosphorylation regulated by mTOR/AAR signaling and CK2 may represent a novel mechanism linking oxygen and nutrient availability to IGF-1 signaling in the decidua.

Glutamine-induced signaling pathways via amino acid receptors in enteroendocrine L cell lines.

Glucagon-like peptide-1 (GLP-1), secreted by gastrointestinal enteroendocrine L cells, induces insulin secretion and is important for glucose homeostasis. GLP-1 secretion is induced by various luminal nutrients, including amino acids. Intracellular Ca2+ and cAMP dynamics play an important role in GLP-1 secretion regulation; however, several aspects of the underlying mechanism of amino acid-induced GLP-1 secretion are not well characterized. We investigated the mechanisms underlying the L-glutamine-induced increase in Ca2+ and cAMP intracellular concentrations ([Ca2+]i and [cAMP]i, respectively) in murine enteroendocrine L cell line GLUTag cells. Application of L-glutamine to cells under low extracellular [Na+] conditions, which inhibited the function of the sodium-coupled L-glutamine transporter, did not induce an increase in [Ca2+]i. Application of G protein-coupled receptor family C group 6 member A and calcium-sensing receptor antagonist showed little effect on [Ca2+]i and [cAMP]i; however, taste receptor type 1 member 3 (TAS1R3) antagonist suppressed the increase in [cAMP]i. To elucidate the function of TAS1R3, which forms a heterodimeric umami receptor with taste receptor type 1 member 1 (TAS1R1), we generated TAS1R1 and TAS1R3 mutant GLUTag cells using the CRISPR/Cas9 system. TAS1R1 mutant GLUTag cells exhibited L-glutamine-induced increase in [cAMP]i, whereas some TAS1R3 mutant GLUTag cells did not exhibit L-glutamine-induced increase in [cAMP]i and GLP-1 secretion. These findings suggest that TAS1R3 is important for L-glutamine-induced increase in [cAMP]i and GLP-1 secretion. Thus, TAS1R3 may be coupled with Gs and related to cAMP regulation.

Expression patterns of L-amino acid receptors in the murine STC-1 enteroendocrine cell line.

Regulation of gut function depends on the detection and response to luminal contents. Luminal L-amino acids (L-AA) are detected by several receptors including metabotropic glutamate receptors 1 and 4 (mGluR1 and mGluR4), calcium-sensing receptor (CaSR), GPRC family C group 6 subtype A receptor (GPRC6A) and umami taste receptor heterodimer T1R1/T1R3. Here, we show that murine mucosal homogenates and STC-1 cells, a murine enteroendocrine cell line, express mRNA for all L-AA receptors. Immunohistochemical analysis demonstrated the presence of all L-AA receptors on STC-1 with CaSR being most commonly expressed and T1R1 least expressed (35% versus 15% of cells); mGluRs and GPRC6a were intermediate (~ 20% of cells). Regarding coexpression of L-AA receptors, the mGluRs and T1R1 were similarly coexpressed with CaSR (10-12% of cells) whereas GPRC6a was coexpressed least (7% of cells). mGluR1 was coexpressed with GPRC6a in 11% of cells whereas coexpression between other receptors was less (2-8% of cells). CaSR and mGluR1 were coexpressed with glucagon-like peptide-1 (GLP-1) and peptide YY (PYY) in 20-25% of cells whereas T1R1 and GPRC6a were coexpressed with GLP-1 and PYY less (8-12% of cells). Only mGluR4 showed differential coexpression with GLP-1 (13%) and PYY (21%). L-Phenylalanine (10 mM) caused a 3-fold increase in GLP-1 release, which was strongly inhibited by siRNA to CaSR indicating functional coupling of CaSR to GLP-1 release. The results suggest that not all STC-1 cells express (and coexpress) L-AA receptors to the same extent and that the pattern of response likely depends on the pattern of expression of L-AA receptors.

Dietary Branched-Chain Amino Acids Regulate Food Intake Partly through Intestinal and Hypothalamic Amino Acid Receptors in Piglets.

Strategies to increase feed intake are of great importance for producing more meat in swine production. Intestinal and hypothalamic amino acid receptors are found to largely participate in feed intake regulation. The purpose of the current research is to study the function of branched-chain amino acid (BCAA) supplementation in the regulation of feed intake through sensors that can detect amino acids in piglets. Twenty-four piglets were assigned one of four treatments and fed one of the experimental diets for either a short period (Expt. 1) or a long period (Expt. 2): a normal protein diet (NP, 20.04% CP), a reduced-protein diet (RP, 17.05% CP), or a reduced-protein test diet supplemented with one of two doses of BCAAs (BCAA1, supplemented with 0.13% l-isoleucine, 0.09% l-leucine, and 0.23% l-valine; BCAA2, supplemented with the 150% standardized ileal digestibility BCAA requirement, as recommended by the National Research Council (2012)). In Expt. 1, no differences were observed in the feed intake among piglets fed different diets ( P > 0.05). In Expt. 2, compared with the RP group, the feed intake of piglets was significantly increased after sufficient BCAAs were supplemented in the BCAA1 group, which was associated with decreased cholecystokinin secretion ( P < 0.05), down-regulated expression of type-1 taste receptors 1/3 (T1R1/T1R3) in the intestine, as well as increased expression of pro-opiomelanocortin, activated general control nonderepressible 2 (GCN2), and eukaryotic initiation factor 2α (eIF2α) in the hypothalamus ( P < 0.05). However, the feed intake was decreased for unknown reasons when the piglets were fed a BCAA over-supplemented diet. Our study confirmed that a BCAA-deficient diet inhibited feed intake through two potential ways: regulating the amino acid T1R1/T1R3 receptor in the intestine or activating GCN2/eIF2α pathways in the hypothalamus.

Cadaverine, a metabolite of the microbiome, reduces breast cancer aggressiveness through trace amino acid receptors.

Recent studies showed that changes to the gut microbiome alters the microbiome-derived metabolome, potentially promoting carcinogenesis in organs that are distal to the gut. In this study, we assessed the relationship between breast cancer and cadaverine biosynthesis. Cadaverine treatment of Balb/c female mice (500 nmol/kg p.o. q.d.) grafted with 4T1 breast cancer cells ameliorated the disease (lower mass and infiltration of the primary tumor, fewer metastases, and lower grade tumors). Cadaverine treatment of breast cancer cell lines corresponding to its serum reference range (100-800 nM) reverted endothelial-to-mesenchymal transition, inhibited cellular movement and invasion, moreover, rendered cells less stem cell-like through reducing mitochondrial oxidation. Trace amino acid receptors (TAARs), namely, TAAR1, TAAR8 and TAAR9 were instrumental in provoking the cadaverine-evoked effects. Early stage breast cancer patients, versus control women, had reduced abundance of the CadA and LdcC genes in fecal DNA, both responsible for bacterial cadaverine production. Moreover, we found low protein expression of E. coli LdcC in the feces of stage 1 breast cancer patients. In addition, higher expression of lysine decarboxylase resulted in a prolonged survival among early-stage breast cancer patients. Taken together, cadaverine production seems to be a regulator of early breast cancer.

Low-Intensity Ultrasound Decreases Ischemia-Induced Edema by Inhibiting N-Methyl-d-Aspartic Acid Receptors.

BACKGROUND: We have previously shown that low-intensity ultrasound (LIUS), a noninvasive mechanical stimulus, inhibits brain edema formation induced by oxygen and glucose deprivation (OGD) or treatment with glutamate, a mediator of OGD-induced edema, in acute rat hippocampal slice model in vitro. METHODS: In this study, we treated the rat hippocampal slices with N-methyl-d-aspartic acid (NMDA) or (S)-3,5-dihydroxyphenylglycine (DHPG) to determine whether these different glutamate receptor agonists induce edema. The hippocampal slices were then either sonicated with LIUS or treated with N-methyl-d-aspartic acid receptor (NMDAR) antagonists, namely, MK-801 and ketamine, and observed their effects on edema formation. RESULTS: We observed that treatment with NMDA, an agonist of ionotropic glutamate receptors, induced brain edema at similar degrees compared with that induced by OGD. However, treatment with DHPG, an agonist of metabotropic glutamate receptors, did not significantly induce brain edema. Treatment with the NMDAR antagonists MK-801 or ketamine efficiently prevented brain edema formation by both OGD and NMDA in a concentration-dependent manner. N-Methyl-d-aspartic acid-induced brain edema was alleviated by LIUS in an intensity-dependent manner when ultrasound was administered at 30, 50, or 100 mW/cm2 for 20 minutes before the induction of the edema. Furthermore, LIUS reduced OGD- and NMDA-induced phosphorylation of NMDARs at Y1325. CONCLUSION: These results suggest that LIUS can inhibit OGD- or NMDA-induced NMDAR activation by preventing NMDAR phosphorylation, thereby reducing a subsequent brain edema formation. The mechanisms by which LIUS inhibits NMDAR phosphorylation need further investigation.

Methionine and valine activate the mammalian target of rapamycin complex 1 pathway through heterodimeric amino acid taste receptor (TAS1R1/TAS1R3) and intracellular Ca2+ in bovine mammary epithelial cells.

Amino acids play a key role in regulating milk protein synthesis partly through activation of the mammalian target of rapamycin (mTOR) signaling pathway. However, the involvement of extracellular AA sensing receptors in this process is not well understood. In nonruminants, it is well established that the AA taste 1 receptor member 1/3 (TAS1R1/TAS1R3) heterodimer contributes to the sensing of most l-AA. Whether this receptor is functional in bovine mammary cells is unknown. The objective of this study was to determine essential AA signaling through TAS1R1/TAS1R3 and their roles in regulating mTOR signaling pathway and casein mRNA abundance in primary bovine mammary epithelial cells and the Mac-T cell line. The bovine mammary epithelial cells were stimulated with complete Dulbecco's modified Eagle's medium (+EAA), medium without EAA (-EAA), or medium supplemented with only 1 of the 10 essential AA, respectively. The nonessential AA levels were the same across all treatments. Small interference RNA targeting TAS1R1 were designed and transfected into bovine primary mammary epithelial cells (bPMEC). Supplementation of a complete mixture of essential AA or Arg, Val, Leu, His, Phe, Met, and Ile individually led to greater mTOR phosphorylation. Phosphorylation of ribosomal protein S6 kinase ß-1 was greater in the presence of Val, Leu, Trp, Met, and Ile. Valine, Leu, Met, and Ile led to greater eIF4E-binding protein 1 phosphorylation. Although +EAA and a few individual AA tested induced increases in intracellular calcium, Met and Val were the most potent. Knockdown of TAS1R1 decreased intracellular calcium in bPMEC cultured with both Val and Met. Phosphorylation of mTOR, ribosomal protein S6 kinase ß-1, and eIF4E-binding protein 1 was lower when TAS1R1 was knocked-down in bPMEC supplemented with Val and Met. In addition, small interference RNA silencing of TAS1R1 resulted in lower ß-casein (CSN2) abundance. The TAS1R1/TAS1R3 receptor may sense extracellular AA and activate mTOR signaling in bovine mammary cells, likely by elevating intracellular calcium concentration. This mechanism appears to have a role in Met- and Val-induced changes in CSN2 mRNA abundance. Further in vivo studies will have to be performed to assess the relevance of this mechanism in the mammary gland.

Overexpression of the leucine-rich receptor-like kinase gene LRK2 increases drought tolerance and tiller number in rice.

Drought represents a key limiting factor of global crop distribution. Receptor-like kinases play major roles in plant development and defence responses against stresses such as drought. In this study, LRK2, which encodes a leucine-rich receptor-like kinase, was cloned and characterized and found to be localized on the plasma membrane in rice. Promoter-GUS analysis revealed strong expression in tiller buds, roots, nodes and anthers. Transgenic plants overexpressing LRK2 exhibited enhanced tolerance to drought stress due to an increased number of lateral roots compared with the wild type at the vegetative stage. Moreover, ectopic expression of LRK2 seedlings resulted in increased tiller development. Yeast two-hybrid screening and bimolecular fluorescence complementation (BiFC) indicated a possible interaction between LRK2 and elongation factor 1 alpha (OsEF1A) in vitro. These results suggest that LRK2 functions as a positive regulator of the drought stress response and tiller development via increased branch development in rice. These findings will aid our understanding of branch regulation in other grasses and support improvements in rice genetics.

Reduced dietary protein level influences the free amino acid and gene expression profiles of selected amino acid transceptors in skeletal muscle of growing pigs.

This study was conducted to evaluate the effect of reduced dietary protein level on growth performance, muscle mass weight, free amino acids (FAA) and gene expression profile of selected amino acid transceptors in different fibre type of skeletal muscle tissues (longissimus dorsi, psoas major, biceps femoris) of growing pigs. A total of 18 cross-bred growing pigs (Large White × Landrace × Duroc) with initial body weight (9.57 ± 0.67 kg) were assigned into three dietary treatments: 20% crude protein (CP) diet (normal recommended, NP), 17% CP diet (low protein, LP) and 14% CP diet (very low protein, VLP). The results indicated improved feed-to-gain ratio was obtained for pigs fed LP and NP diets (p < 0.01), while the pigs fed VLP diet showed the worst growth performance (p < 0.01). There was no significant difference in the weights of longissimus dorsi and psoas major muscle between LP and NP groups (p > 0.05). Majority of the determined FAA concentration of LP group were greater than or equal to those of NP group in both longissimus dorsi and psoas major muscle (p < 0.01). Further, the mRNA expression levels of sodium-coupled neutral amino acid transceptor 2, L-type amino acid transceptor 1 and proton-assisted amino acid transceptors 2 were higher in skeletal muscle tissue in LP group compared to those of the pigs fed NP or VLP diet. These results suggested that reduced dietary protein level (3 points of percentage less than recommended level) would upregulate the mRNA expression of amino acid transceptors to enhance the absorption of FAA in skeletal muscle of growing pigs. There seems to be a relationship between response of AA transceptors to the dietary protein level in skeletal muscle tissue of different fibre type. To illustrate the underlying mechanisms will be beneficial to animal nutrition.

Structural Analysis of the Ligand-Binding Domain of the Aspartate Receptor Tar from Escherichia coli.

The Escherichia coli cell-surface aspartate receptor Tar mediates bacterial chemotaxis toward an attractant, aspartate (Asp), and away from a repellent, Ni(2+). These signals are transmitted from the extracellular region of Tar to the cytoplasmic region via the transmembrane domain. The mechanism by which extracellular signals are transmitted into the cell through conformational changes in Tar is predicted to involve a piston displacement of one of the α4 helices of the homodimer. To understand the molecular mechanisms underlying the induction of Tar activity by an attractant, the three-dimensional structures of the E. coli Tar periplasmic domain with and without bound aspartate, Asp-Tar and apo-Tar, respectively, were determined. Of the two ligand-binding sites, only one site was occupied, and it clearly showed the electron density of an aspartate. The slight changes in conformation and the electrostatic surface potential around the aspartate-binding site were observed. In addition, the presence of an aspartate stabilized residues Phe-150' and Arg-73. A pistonlike displacement of helix α4b' was also induced by aspartate binding as predicted by the piston model. Taken together, these small changes might be related to the induction of Tar activity and might disturb binding of the second aspartate to the second binding site in E. coli.

The crystal structure of the tandem-PAS sensing domain of Campylobacter jejuni chemoreceptor Tlp1 suggests indirect mechanism of ligand recognition.

Chemotaxis and motility play an important role in the colonisation of avian and human hosts by Campylobacter jejuni. Chemotactic recognition of extracellular signals is mediated by the periplasmic sensing domain of methyl-accepting chemotactic proteins (membrane-embedded receptors). In this work, we report a high-resolution structure of the periplasmic sensing domain of transducer-like protein 1 (Tlp1), an aspartate receptor of C. jejuni. Crystallographic analysis revealed that it contains two Per-Arnt-Sim (PAS) subdomains. An acetate and chloride ions (both from the crystallisation buffer) were observed bound to the membrane-proximal and membrane-distal PAS subdomains, respectively. Surprisingly, despite being crystallised in the presence of aspartate, the structure did not show any electron density corresponding to this amino acid. Furthermore, no binding between the sensing domain of Tlp1 and aspartate was detected by microcalorimetric experiments. These structural and biophysical data suggest that Tlp1 does not sense aspartate directly; instead, ligand recognition is likely to occur indirectly via an as yet unidentified periplasmic binding protein.

Identification of a Vibrio cholerae chemoreceptor that senses taurine and amino acids as attractants.

Vibrio cholerae, the etiological agent of cholera, was found to be attracted by taurine (2-aminoethanesulfonic acid), a major constituent of human bile. Mlp37, the closest homolog of the previously identified amino acid chemoreceptor Mlp24, was found to mediate taxis to taurine as well as L-serine, L-alanine, L-arginine, and other amino acids. Methylation of Mlp37 was enhanced upon the addition of taurine and amino acids. Isothermal titration calorimetry demonstrated that a purified periplasmic fragment of Mlp37 binds directly to taurine, L-serine, L-alanine and L-arginine. Crystal structures of the periplamic domain of Mlp37 revealed that L-serine and taurine bind to the membrane-distal PAS domain in essentially in the same way. The structural information was supported by characterising the in vivo properties of alanine-substituted mutant forms of Mlp37. The fact that the ligand-binding domain of the L-serine complex had a small opening, which would accommodate a larger R group, accounts for the broad ligand specificity of Mlp37 and allowed us to visualise ligand binding to Mlp37 with fluorescently labelled L-serine. Taken together, we conclude that Mlp37 serves as the major chemoreceptor for taurine and various amino acids.

All trans retinoic acid (ATRA) mediated modulation of N-methyl D-aspartate receptor (NMDAR) and Kruppel like factor 11 (KLF11) expressions in the mitigation of ethanol induced alterations in the brain.

BACKGROUND: Damaging effects that chronic ethanol exposure causes to the brain and the neurons are well documented. Ethanol and its toxic metabolites increase the oxidative stress in brain. Chronic exposure to ethanol leads to upregulation of N-methyl D-aspartate receptors (NMDAR) and also activates Kruppel like factor 11 (KLF11) mediated death cascade and thereby neurodegeneration. OBJECTIVE: Ethanol depletes vitamin A stores. But supplementation of vitamin A exacerbates ethanol induced toxicity since alcohol and its metabolites are competitive inhibitors of the enzymes involved in the metabolism of vitamin A. Hence, in this study we investigated the impact of co-administration of ethanol and all trans retinoic acid (ATRA), active metabolite of vitamin A, on ethanol induced alterations to the brain. MATERIALS AND METHODS: Male Sprague Dawley rats, adolescent, were grouped as follows and maintained for 90 days. I - Control, II - Ethanol (4 g/kg b.w.), III - ATRA (100 µg/kg b.w.), IV - Ethanol (4 g/kg b.w.), +ATRA (100 µg/kg b.w.). Oxidative stress and the mRNA expression of various receptors for the neurotransmitter involved in glutamergic, serotonergic and gabaergic pathways were studied in the brain homogenate. RESULTS: Ethanol treatment was shown to decrease brain weight and it was increased on ATRA treatment. Increase in oxidative stress due to ethanol treatment was also brought down on ATRA administration. Ethanol induced upregulation of NMDAR and KLF11 was also downregulated on ATRA supplementation. The alterations in the levels of neurotransmitters and the expression of their receptors due to ethanol treatment also were ameliorated on ATRA supplementation. CONCLUSION: Our results show that ATRA supplementation mitigates the ethanol induced alterations in the brain by reducing oxidative stress in the brain with concurrent suppression of NMDAR and KLF11 expression leading to enhanced catabolism of neurotransmitters.

Immunogold cytochemistry in neuroscience.

The complexity of the central nervous system calls for immunocytochemical procedures that allow target proteins to be localized with high precision and with opportunities for quantitation. Immunogold procedures stand out as particularly powerful in this regard. Although these procedures have found wide application in the neuroscience community, they present limitations and pitfalls that must be taken into account. At the same time, these procedures offer potentials that remain to be fully realized.

Astroglial amino acid-based transmitter receptors.

Amino acids appear in prebiotic period being one of the first organic molecules on Earth. For neurobiologists, it is of importance that AAs are not only representing building blocks of life, but are also the essential part of metabolism and cellular signaling. In the mammalian brain, the most common excitatory and inhibitory transmitters acting upon cellular plasmalemmal receptors are the amino acid glutamate and its derivative γ-aminobutyric acid, respectively. Other amino acids, i.e. aspartate, glycine, D-serine, and homocysteic acid, as well as the sulfonic acid taurine, are also active compounds involved in receptor-mediated brain signaling. Receptors for these amino acid-based transmitters are either ion channels, also referred to as ionotropic receptors, or metabotropic, i.e. seven transmembrane domain G-protein coupled receptors. In this mini-review, we focus our interest on amino acid-based transmitter receptors on neuroglia, astrocytes in particular.

Ligand affinity and kinase activity are independent of bacterial chemotaxis receptor concentration: insight into signaling mechanisms.

Binding of attractant to bacterial chemotaxis receptors initiates a transmembrane signal that inhibits the kinase CheA bound ~300 Å distant at the other end of the receptor. Chemoreceptors form large clusters in many bacterial species, and the extent of clustering has been reported to vary with signaling state. To test whether ligand binding regulates kinase activity by modulating a clustering equilibrium, we measured the effects of two-dimensional receptor concentration on kinase activity in proteoliposomes containing the purified Escherichia coli serine receptor reconstituted into vesicles over a range of lipid:protein molar ratios. The IC(50) of kinase inhibition was unchanged despite a 10-fold change in receptor concentration. Such a change in concentration would have produced a measurable shift in the IC(50) if receptor clustering were involved in kinase regulation, based on a simple model in which the receptor oligomerization and ligand binding equilibria are coupled. These results indicate that the primary signal, ligand control of kinase activity, does not involve a change in receptor oligomerization state. In combination with previous work on cytoplasmic fragments assembled on vesicle surfaces [Besschetnova, T. Y., et al. (2008) Proc. Natl. Acad. Sci. U.S.A.105, 12289-12294], this suggests that binding of ligand to chemotaxis receptors inhibits the kinase by inducing a conformational change that expands the membrane area occupied by the receptor cytoplasmic domain, without changing the number of associated receptors in the signaling complex.

Amino acid sensing mechanisms: an Achilles heel in cancer?

The act of increasing mass, either in non-dividing cells or in dividing cells seeking to provide new material for daughter cells, depends upon the continued presence of extracellular nutrients in order to conserve mass. For amino acid nutrients, it appears that their insufficiency for new protein synthesis is actively monitored by both prokaryotic and eukaryotic cells, eliciting appropriate cellular responses that may depend not only on bulk nutrient supply, but also on the abundance of specific amino acids.

Cell-selective labeling of bacterial proteomes with an orthogonal phenylalanine amino acid reporter.

Orthogonal amino acid reporters allow the selective labeling of different cell types in heterogeneous populations through the expression of engineered aminoacyl tRNA synthetases. Here, we demonstrate that para-ethynylphenylalanine (PEP) can be used as an orthogonal amino acid reporter for efficient selective labeling of an intracellular bacterial pathogen during infection.