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Dual binding mode of "bitter sugars" to their human bitter taste receptor target.

ABSTRACT: The 25 human bitter taste receptors (hTAS2Rs) are responsible for detecting bitter molecules present in food, and they also play several physiological and pathological roles in extraoral compartments. Therefore, understanding their ligand specificity is important both for food research and for pharmacological applications. Here we provide a molecular insight into the exquisite molecular recognition of bitter ?-glycopyranosides by one of the members of this receptor subclass, hTAS2R16. Most of its agonists have in common the presence of a ?-glycopyranose unit along with an extremely structurally diverse aglycon moiety. This poses the question of how hTAS2R16 can recognize such a large number of "bitter sugars". By means of hybrid molecular mechanics/coarse grained molecular dynamics simulations, here we show that the three hTAS2R16 agonists salicin, arbutin and phenyl-?-D-glucopyranoside interact with the receptor through a previously unrecognized dual binding mode. Such mechanism may offer a seamless way to fit different aglycons inside the binding cavity, while maintaining the sugar bound, similar to the strategy used by several carbohydrate-binding lectins. Our prediction is validated a posteriori by comparison with mutagenesis data and also rationalizes a wealth of structure-activity relationship data. Therefore, our findings not only provide a deeper molecular characterization of the binding determinants for the three ligands studied here, but also give insights applicable to other hTAS2R16 agonists. Together with our results for other hTAS2Rs, this study paves the way to improve our overall understanding of the structural determinants of ligand specificity in bitter taste receptors.

SUBMITTER: Fierro F

PROVIDER: S-EPMC6560132 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Dual binding mode of "bitter sugars" to their human bitter taste receptor target.

Fierro Fabrizio F Giorgetti Alejandro A Carloni Paolo P Meyerhof Wolfgang W Alfonso-Prieto Mercedes M

Scientific reports 20190611 1

The 25 human bitter taste receptors (hTAS2Rs) are responsible for detecting bitter molecules present in food, and they also play several physiological and pathological roles in extraoral compartments. Therefore, understanding their ligand specificity is important both for food research and for pharmacological applications. Here we provide a molecular insight into the exquisite molecular recognition of bitter β-glycopyranosides by one of the members of this receptor subclass, hTAS2R16. Most of it ...[more]

PMID: 31186454

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Project description:BackgroundAlleles of the receptor gene TAS2R38 are responsible in part for the variation in bitter taste perception of 6-n-propylthiouracil (PROP) and structurally similar compounds (eg, glucosinolates in cruciferous vegetables). At low concentrations, people with the PAV ("taster" amino acid sequence) form of TAS2R38 perceive these bitter compounds, whereas most with the AVI ("nontaster" amino acid sequence) form do not; heterozygotes (PAV/AVI) show the widest range of bitter perception.ObjectivesThe objectives were to examine individual differences in expression of PAV-TAS2R38 messenger RNA (mRNA) among heterozygotes, to test the hypotheses that the abundance of allele-specific gene expression accounts for the variation in human bitter taste perception, and to relate to dietary intake of bitter-tasting beverages and foods.DesignHeterozygous individuals (n = 22) provided psychophysical evaluation of the bitterness of PROP, glucosinolate-containing broccoli juice, non-glucosinolate-containing carrot juice, and several bitter non-TAS2R38 ligands as well as dietary recalls. Fungiform taste papillae were examined for allele-specific TAS2R38 expression by using quantitative polymerase chain reaction.ResultsPAV-TAS2R38 mRNA expression was measured in 18 of 22 heterozygous subjects. Relative expression varied widely and positively correlated with ratings of bitterness intensity of PROP (P = 0.007) and broccoli juice (P = 0.004) but not of the control solutions carrot juice (P = 0.26), NaCl (P = 0.68), caffeine (P = 0.24), or urea (P = 0.47). Expression amounts were related to self-reported recent and habitual caffeine intake (P = 0.060, P = 0.005); vegetable intake was too low to analyze.ConclusionsWe provide evidence that PAV-TAS2R38 expression amount correlates with individual differences in bitter sensory perception and diet. The nature of this correlation calls for additional research on the molecular mechanisms associated with some individual differences in taste perception and food intake. The trial was registered at clinicaltrials.gov as NCT01399944.

Project description:Bitter taste stimuli are detected by a diverse family of G protein-coupled receptors (GPCRs) expressed in gustatory cells. Each bitter taste receptor (TAS2R) responds to an array of compounds, many of which are toxic and can be found in nature. For example, human TAS2R16 (hTAS2R16) responds to ?-glucosides such as salicin, and hTAS2R38 responds to thiourea-containing molecules such as glucosinolates and phenylthiocarbamide (PTC). While many substances are known to activate TAS2Rs, only one inhibitor that specifically blocks bitter receptor activation has been described. Here, we describe a new inhibitor of bitter taste receptors, p-(dipropylsulfamoyl)benzoic acid (probenecid), that acts on a subset of TAS2Rs and inhibits through a novel, allosteric mechanism of action. Probenecid is an FDA-approved inhibitor of the Multidrug Resistance Protein 1 (MRP1) transporter and is clinically used to treat gout in humans. Probenecid is also commonly used to enhance cellular signals in GPCR calcium mobilization assays. We show that probenecid specifically inhibits the cellular response mediated by the bitter taste receptor hTAS2R16 and provide molecular and pharmacological evidence for direct interaction with this GPCR using a non-competitive (allosteric) mechanism. Through a comprehensive analysis of hTAS2R16 point mutants, we define amino acid residues involved in the probenecid interaction that result in decreased sensitivity to probenecid while maintaining normal responses to salicin. Probenecid inhibits hTAS2R16, hTAS2R38, and hTAS2R43, but does not inhibit the bitter receptor hTAS2R31 or non-TAS2R GPCRs. Additionally, structurally unrelated MRP1 inhibitors, such as indomethacin, fail to inhibit hTAS2R16 function. Finally, we demonstrate that the inhibitory activity of probenecid in cellular experiments translates to inhibition of bitter taste perception of salicin in humans. This work identifies probenecid as a pharmacological tool for understanding the cell biology of bitter taste and as a lead for the development of broad specificity bitter blockers to improve nutrition and medical compliance.

Project description:The human G-protein-coupled bitter taste receptor T2R38 has recently been demonstrated to be expressed on peripheral blood neutrophils, monocytes and lymphocytes. To further define a potential contribution of the T2R38 receptor in adaptive immune response, the objective of this study was to analyze its expression in resting and activated lymphocytes and T cell subpopulations. Freshly isolated PBMC from healthy donors were used for expression analysis by flow cytometry. Quantum™ MESF beads were applied for quantification in absolute fluorescence units. Activation methods of T cells were anti-CD3/CD28, phytohaemagglutinin (PHA) or phorbol 12-myristate 13-acetate (PMA) together with ionomycin. Lymphocytes from young donors expressed higher levels of T2R38 compared to the elderly. CD3+ T cells expressed higher levels that CD19+ B cells. Receptor expression followed T cell activation with an upregulation within 24 h and a peak at 72 h. Higher levels of T2R38 were produced in lymphocytes by stimulation with anti-CD3/CD28 compared to PHA or PMA/ionomycin. Both subpopulations of CD4+ as well as CD8+ T cells were found to express the T2R38 receptor; this was higher in CD4+ than CD8+ cells; the amount of T2R38 in central and effector memory cells was higher as compared to naïve cells, although this was not statistically significant for CD8+ cells without prior activation by anti-CD3/CD28. Upon treatment of PBMC with the natural T2R38 agonist goitrin Calcium flux was activated in the lymphocyte population with functional T2R38 receptor at >20 ?M which was completely blocked by phospholipase C?-2 inhibitor U73211. Further, goitrin selectively inhibited TNF-alpha secretion in PBMC with functional T2R38. This quantitative analysis of T2R38 expression in distinct PBMC subsets may provide a basis for understanding the significance of bitter compounds in immune modulation. Whether these findings can have implications for the treatment of inflammatory and immunologic disorders by bitter tasting pharmaceuticals or foods needs further investigation.

Project description:Solitary chemosensory cells (SCCs) are specialized cells in the respiratory epithelium that respond to noxious chemicals including bacterial signaling molecules. SCCs express components of bitter taste transduction including the taste receptor type 2 (TAS2R) bitter taste receptors and downstream signaling effectors: ?-Gustducin, phospholipase C?2 (PLC?2), and transient receptor potential cation channel subfamily M member 5 (TRPM5). When activated, SCCs evoke neurogenic reflexes, resulting in local inflammation. The purpose of this study was to test for the presence SCCs in human sinonasal epithelium, and to test for a correlation with inflammatory disease processes such as allergic rhinitis and chronic rhinosinusitis.Patient demographics and biopsies of human sinonasal mucosa were obtained from control patients (n = 7) and those with allergic rhinitis and/or chronic rhinosinusitis (n = 15). Reverse transcription polymerase chain reaction (RT-PCR), quantitative PCR (qPCR), and immunohistochemistry were used to determine whether expression of signaling effectors was altered in diseased patients.RT-PCR demonstrated that bitter taste receptors TAS2R4, TAS2R14, and TAS2R46, and downstream signaling effectors ?-Gustducin, PLC?2, and TRPM5 are expressed in the inferior turbinate, middle turbinate, septum, and uncinate of both control and diseased patients. PLC?2/TRPM5-immunoreactive SCCs were identified in the sinonasal mucosa of both control and diseased patients. qPCR showed similar expression of ?-Gustducin and TRPM5 in the uncinate process of control and diseased groups, and there was no correlation between level of expression and 22-item Sino-Nasal Outcomes Test (SNOT-22) or pain scores.SCCs are present in human sinonasal mucosa in functionally relevant areas. Expression level of signaling effectors was similar in control and diseased patients and did not correlate with measures of pain and inflammation. Further study into these pathways may provide insight into nasal inflammatory diseases and may offer potential therapeutic targets.

Dataset Information

Dual binding mode of "bitter sugars" to their human bitter taste receptor target.

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Dual binding mode of "bitter sugars" to their human bitter taste receptor target.

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