Snooker Structure-Based Pharmacophore Model Explains Differences in Agonist and Blocker Binding to Bitter Receptor hTAS2R39

The human bitter taste receptor hTAS2R39 can be activated by many dietary (iso)flavonoids. Furthermore, hTAS2R39 activity can be blocked by 6-methoxyflavanones, 4’-fluoro-6-methoxyflavanone in particular. A structure-based pharmacophore model of the hTAS2R39 binding pocket was built using Snooker software, which has been used successfully before for drug design of GPCRs of the rhodopsin subfamily. For the validation of the model, two sets of compounds, both of which contained actives and inactives, were used: (i) an (iso)flavonoid-dedicated set, and (ii) a more generic, structurally diverse set. Agonists were characterized by their linear binding geometry and the fact that they bound deeply in the hTAS2R39 pocket, mapping the hydrogen donor feature based on T5.45 and N3.36, analogues of which have been proposed to play a key role in activation of GPCRs. Blockers lack hydrogen-bond donors enabling contact to the receptor. Furthermore, they had a crooked geometry, which could sterically hinder movement of the TM domains upon receptor activation. Our results reveal characteristics of hTAS2R39 agonist and bitter blocker binding, which might facilitate the development of blockers suitable to counter the bitterness of dietary hTAS2R39 agonists in food applications.     

Human Bitter Taste Receptors hTAS2R8 and hTAS2R39 with Differential Functions to Recognize Bitter Peptides

The strong bitter peptide, Phe-Phe-Pro-Arg, activated cultured cells expressing either of the known human bitter taste receptors, hTAS2R8 and hTAS2R39. The partial structure of Pro-Arg activated hTAS2R39, but did not activate hTAS2R8. These receptors may not indiscriminately recognize bitter peptides, but have a differential function in their recognition.     

Human bitter taste receptors hTAS2R8 and hTAS2R39 with differential functions to recognize bitter peptides

The strong bitter peptide, Phe-Phe-Pro-Arg, activated cultured cells expressing either of the known human bitter taste receptors, hTAS2R8 and hTAS2R39. The partial structure of Pro-Arg activated hTAS2R39, but did not activate hTAS2R8. These receptors may not indiscriminately recognize bitter peptides, but have a differential function in their recognition.     

Activation of human bitter taste receptors by polymethoxylated flavonoids

Tangeretin and nobiletin are polymethoxylated flavonoids in citrus peel. Both tangeretin and nobiletin are bitter; however, their bitterness has not been evaluated using human bitter taste receptors (hTAS2Rs). We screened 25 kinds of hTAS2Rs and found that hTAS2R14 and hTAS2R46 received both compounds.     

Probenecid inhibits the human bitter taste receptor TAS2R16 and suppresses bitter perception of salicin

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 β-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.     

The human taste receptor hTAS2R14 responds to a variety of different bitter compounds

The recent advances in the functional expression of TAS2Rs in heterologous systems resulted in the identification of bitter tastants that specifically activate receptors of this family. All bitter taste receptors reported to date exhibit a pronounced selectivity for single substances or structurally related bitter compounds. In the present study we demonstrate the expression of the hTAS2R14 gene by RT-PCR analyses and in situ hybridisation in human circumvallate papillae. By functional expression in HEK-293T cells we show that hTAS2R14 displays a, so far, unique broad tuning towards a variety of structurally diverse bitter compounds, including the potent neurotoxins, (-)-alpha-thujone, the pharmacologically active component of absinthe, and picrotoxinin, a poisonous substance of fishberries. The observed activation of heterologously expressed hTAS2R14 by low concentrations of (-)-alpha-thujone and picrotoxinin suggests that the receptor is sufficiently sensitive to caution us against the ingestion of toxic amounts of these substances.     

Bitter peptides activate hTAS2Rs, the human bitter receptors

Fermented food contains numerous peptides derived from material proteins. Bitter peptides formed during the fermentation process are responsible for the bitter taste of fermented food. We investigated whether human bitter receptors (hTAS2Rs) recognize bitterness of peptides with a heterologous expression system. HEK293 cells expressing hTAS2R1, hTAS2R4, hTAS2R14, and hTAS2R16 responded to bitter casein digests. Among those cells, the hTAS2R1-expressing cell was most strongly activated by the synthesized bitter peptides Gly-Phe and Gly-Leu, and none of the cells was activated by the non-bitter dipeptide Gly-Gly. The results showed that these bitter peptides, as well as many other bitter compounds, activate hTAS2Rs, suggesting that humans utilize these hTAS2Rs to recognize and perceive the structure and bitterness of peptides.     

Insights into the Binding of Phenyltiocarbamide (PTC) Agonist to Its Target Human TAS2R38 Bitter Receptor

Humans'' bitter taste perception is mediated by the hTAS2R subfamily of the G protein-coupled membrane receptors (GPCRs). Structural information on these receptors is currently limited. Here we identify residues involved in the binding of phenylthiocarbamide (PTC) and in receptor activation in one of the most widely studied hTAS2Rs (hTAS2R38) by means of structural bioinformatics and molecular docking. The predictions are validated by site-directed mutagenesis experiments that involve specific residues located in the putative binding site and trans-membrane (TM) helices 6 and 7 putatively involved in receptor activation. Based on our measurements, we suggest that (i) residue N103 participates actively in PTC binding, in line with previous computational studies. (ii) W99, M100 and S259 contribute to define the size and shape of the binding cavity. (iii) W99 and M100, along with F255 and V296, play a key role for receptor activation, providing insights on bitter taste receptor activation not emerging from the previously reported computational models.     

Evaluation of the bitterness of green tea catechins by a cell-based assay with the human bitter taste receptor hTAS2R39

Catechins have a broad range of physiological functions and act as the main taste ingredient of green tea. Although catechins show a strong bitterness, the bitter taste receptor for catechins has not been fully understood. The objective of this study was to identify the receptor for the major green tea catechins such as (−)-epicatechin (EC), (−)-epicatechin gallate (ECg), (−)-epigallocatechin (EGC), and (−)-epigallocatechin gallate (EGCg). By the cell-based assay using cultured cells expressing human bitter taste receptor, a clear response of hTAS2R39-expressing cells was observed to 300 μM of either ECg or EGCg, which elicit a strong bitterness in humans. The response of hTAS2R39-expressing cells to ECg was the strongest among the tested catechins, followed by EGCg. Because the cellular response to EC and EGC is much weaker than those of ECg and EGCg, galloyl groups was strongly supposed to be involved in the bitter intensity. This finding is similar to the observations of taste intensity obtained from a human sensory study. Our results suggest the participation of hTAS2R39 in the detection of catechins in humans, indicating the possibility that bitterness of tea catechins can be evaluated by using cells expressing hTAS2R39.     

Genetic variation in the hTAS2R38 taste receptor and food consumption among Finnish adults

Genetic variation in bitter taste receptors, such as hTAS2R38, may affect food preferences and intake. The aim of the present study was to investigate the association between bitter taste receptor haplotypes and the consumption of vegetables, fruits, berries and sweet foods among an adult Finnish population. A cross-sectional design utilizing data from the Cardiovascular Risk in Young Finns cohort from 2007, which consisted of 1,903 men and women who were 30–45 years of age from five different regions in Finland, was employed. DNA was extracted from blood samples, and hTAS2R38 polymorphisms were determined based on three SNPs (rs713598, rs1726866 and rs10246939). Food consumption was assessed with a validated food frequency questionnaire. The prevalence of the bitter taste-sensitive (PAV/PAV) haplotype was 11.3 % and that of the insensitive (AVI/AVI) haplotype was 39.5 % among this Finnish population. PAV homozygotic women consumed fewer vegetables than did the AVI homozygotic women, 269 g/day (SD 131) versus 301 g/day (SD 187), respectively, p = 0.03 (multivariate ANOVA). Furthermore, the intake of sweet foods was higher among the PAV homozygotes of both genders. Fruit and berry consumption did not differ significantly between the haplotypes in either gender. Individuals perceive foods differently, and this may influence their patterns of food consumption. This study showed that the hTAS2R38 taste receptor gene variation was associated with vegetable and sweet food consumption among adults in a Finnish population.     

Fluorescence-based optimization of human bitter taste receptor expression in Saccharomyces cerevisiae

Human TAS2 receptors (hTAS2Rs) perceive bitter tastants, but few studies have explored the structure-function relationships of these receptors. In this paper, we report our trials on the large-scale preparations of hTAS2Rs for structural analysis. Twenty-five hTAS2Rs were expressed using a GFP-fusion yeast system in which the constructs and the culture conditions (e.g., the signal sequence, incubation time and temperature after induction) were optimized by measuring GFP fluorescence. After optimization, five hTAS2Rs (hTAS2R7, hTAS2R8, hTAS2R16, hTAS2R41, and hTAS2R48) were expressed at levels greater than 1 mg protein/L of culture, which is a preferable level for purification and crystallization. Among these five bitter taste receptors, hTAS2R41 exhibited the highest detergent solubilization efficiency of 87.1% in n-dodecyl-β-d-maltopyranoside (DDM)/cholesteryl hemisuccinate (CHS). Fluorescence size-exclusion chromatography showed that hTAS2R41 exhibited monodispersity in DDM/CHS without aggregates, suggesting that hTAS2R41 is a good target for future crystallization trials.     

Characterization of the ��-d-Glucopyranoside Binding Site of the Human Bitter Taste Receptor hTAS2R16

G-protein-coupled receptors mediate the senses of taste, smell, and vision in mammals. Humans recognize thousands of compounds as bitter, and this response is mediated by the hTAS2R family, which is one of the G-protein-coupled receptors composed of only 25 receptors. However, structural information on these receptors is limited. To address the molecular basis of bitter tastant discrimination by the hTAS2Rs, we performed ligand docking simulation and functional analysis using a series of point mutants of hTAS2R16 to identify its binding sites. The docking simulation predicted two candidate binding structures for a salicin-hTAS2R16 complex, and at least seven amino acid residues in transmembrane 3 (TM3), TM5, and TM6 were shown to be involved in ligand recognition. We also identified the probable salicin-hTAS2R16 binding mode using a mutated receptor experiment. This study characterizes the molecular interaction between hTAS2R16 and β-d-glucopyranoside and will also facilitate rational design of bitter blockers.     

Functions of human bitter taste receptors depend on N-glycosylation

Human bitter taste receptors of the TAS2R gene family play a crucial role as warning sensors against the ingestion of toxic food compounds. Moreover, the genetically highly polymorphic hTAS2Rs recognize an enormous number of structurally diverse toxic and non-toxic bitter substances, and hence, may substantially influence our individual eating habits. Heterologous expression in mammalian cells is a useful tool to investigate interactions between these receptors and their agonists. However, many bitter taste receptors are poorly expressed at the cell surface of heterologous cells requiring the addition of plasma membrane export promoting epitopes to the native receptor proteins. Currently, nothing is known about amino acid motifs or other receptor-intrinsic features of TAS2Rs affecting plasma membrane association. In the present study, we analyzed the Asn-linked glycosylation of hTAS2Rs at a consensus sequence in the second extracellular loop, which is conserved among all 25 hTAS2Rs. Non-glycosylated receptors exhibit substantially lower cell surface localization and reduced association with the cellular chaperone calnexin. As the auxiliary factors receptor transporting proteins 3 and 4 are able to restore the function of non-glycosylated hTAS2R16 partially, we conclude that glycosylation is important for receptor maturation but not for its function per se .     

Computational Studies of Ligand-Receptor Interactions in Bitter Taste Receptors

Phenylthiocarbamide tastes intensely bitter to some individuals, but others find it completely tasteless. Recently, it was suggested that phenylthiocarbamide elicits bitter taste by interacting with a human G protein-coupled receptor (hTAS2R38) encoded by the PTC gene. The phenylthiocarbamide nontaster trait was linked to three single nucleotide polymorphisms occurring in the PTC gene. Using the crystal structure of bovine rhodopsin as template, we generated the 3D structure of hTAS2R38 bitter taste receptor. We were able to map on the receptor structure the amino acids affected by the genetic polymorphisms and to propose molecular functions for two of them that explained the emergence of the nontaster trait. We used molecular docking simulations to find that phenylthiocarbamide exhibited a higher affinity for the target receptor than the structurally similar molecule 6-n-propylthiouracil, in line with recent experimental studies. A 3D model was constructed for the hTAS2R16 bitter taste receptor as well, by applying the same protocol. We found that the recently published experimental ligand binding affinity data for this receptor correlated well with the binding scores obtained from our molecular docking calculations.     

Computational studies of ligand-receptor interactions in bitter taste receptors

Phenylthiocarbamide tastes intensely bitter to some individuals, but others find it completely tasteless. Recently, it was suggested that phenylthiocarbamide elicits bitter taste by interacting with a human G protein-coupled receptor (hTAS2R38) encoded by the PTC gene. The phenylthiocarbamide nontaster trait was linked to three single nucleotide polymorphisms occurring in the PTC gene. Using the crystal structure of bovine rhodopsin as template, we generated the 3D structure of hTAS2R38 bitter taste receptor. We were able to map on the receptor structure the amino acids affected by the genetic polymorphisms and to propose molecular functions for two of them that explained the emergence of the nontaster trait. We used molecular docking simulations to find that phenylthiocarbamide exhibited a higher affinity for the target receptor than the structurally similar molecule 6-n-propylthiouracil, in line with recent experimental studies. A 3D model was constructed for the hTAS2R16 bitter taste receptor as well, by applying the same protocol. We found that the recently published experimental ligand binding affinity data for this receptor correlated well with the binding scores obtained from our molecular docking calculations.     

The molecular basis of individual differences in phenylthiocarbamide and propylthiouracil bitterness perception

Individual differences in perception are ubiquitous within the chemical senses: taste, smell, and chemical somesthesis . A hypothesis of this fact states that polymorphisms in human sensory receptor genes could alter perception by coding for functionally distinct receptor types . We have previously reported evidence that sequence variants in a presumptive bitter receptor gene (hTAS2R38) correlate with differences in bitterness recognition of phenylthiocarbamide (PTC) . Here, we map individual psychogenomic pathways for bitter taste by testing people with a variety of psychophysical tasks and linking their individual perceptions of the compounds PTC and propylthiouracil (PROP) to the in vitro responses of their TAS2R38 receptor variants. Functional expression studies demonstrate that five different haplotypes from the hTAS2R38 gene code for operatively distinct receptors. The responses of the three haplotypes we also tested in vivo correlate strongly with individuals' psychophysical bitter sensitivities to a family of compounds. These data provide a direct molecular link between heritable variability in bitter taste perception to functional variations of a single G protein coupled receptor that responds to compounds such as PTC and PROP that contain the N-C=S moiety. The molecular mechanisms of perceived bitterness variability have therapeutic implications, such as helping patients to consume beneficial bitter-tasting compounds-for example, pharmaceuticals and selected phytochemicals.     

Flavonoids from the seeds of Lonchocarpus costaricensis

Seven flavonoids have been isolated from the seeds of Lonchocarpus costaricensis of which four appear to be novel. The new compounds have been identified as the β-hydroxychalcone demethylpraecansone-B and the flavanones 7-(3,3-dimethylallyloxy)-8-(3-hydroxy-3-methyl-trans-but-1-enyl)flavanone, 7-(3,3-dimethylallyloxy)-8-(3,3-dimethylallyl)-5-methoxyflavanone and 8-(3,3-dimethylallyl)-5,7-dimethoxyflavanone.     

Flower colour and cytochromes P450

Cytochromes P450 play important roles in biosynthesis of flavonoids and their coloured class of compounds, anthocyanins, both of which are major floral pigments. The number of hydroxyl groups on the B-ring of anthocyanidins (the chromophores and precursors of anthocyanins) impact the anthocyanin colour, the more the bluer. The hydroxylation pattern is determined by two cytochromes P450, flavonoid 3′-hydroxylase (F3′H) and flavonoid 3′,5′-hydroxylase (F3′5′H) and thus they play a crucial role in the determination of flower colour. F3′H and F3′5′H mostly belong to CYP75B and CYP75A, respectively, except for the F3′5′Hs in Compositae that were derived from gene duplication of CYP75B and neofunctionalization. Roses and carnations lack blue/violet flower colours owing to the deficiency of F3′5′H and therefore lack the B-ring-trihydroxylated anthocyanins based upon delphinidin. Successful redirection of the anthocyanin biosynthesis pathway to delphinidin was achieved by expressing F3′5′H coding regions resulting in carnations and roses with novel blue hues that have been commercialized. Suppression of F3′5′H and F3′H in delphinidin-producing plants reduced the number of hydroxyl groups on the anthocyanidin B-ring resulting in the production of monohydroxylated anthocyanins based on pelargonidin with a shift in flower colour to orange/red. Pelargonidin biosynthesis is enhanced by additional expression of a dihydroflavonol 4-reductase that can use the monohydroxylated dihydrokaempferol (the pelargonidin precursor). Flavone synthase II (FNSII)-catalysing flavone biosynthesis from flavanones is also a P450 (CYP93B) and contributes to flower colour, because flavones act as co-pigments to anthocyanins and can cause blueing and darkening of colour. However, transgenic plants expression of a FNSII gene yielded paler flowers owing to a reduction of anthocyanins because flavanones are precursors of anthocyanins and flavones.     

Structure-function relationships of the human bitter taste receptor hTAS2R1: insights from molecular modeling studies

Bitter taste receptors (T2Rs) belong to G-protein-coupled receptors (GPCRs). Despite extensive studies, the precise mechanisms of GPCR activation are still poorly understood. In this study, the models of the human bitter taste receptor hTAS2R1 alone and in complex with various ligands were constructed on the basis of template-based modeling and molecular docking. Then these models were subjected to all-atom molecular dynamics (MD) simulations in explicit lipid bilayers. The binding pocket of hTAS2R1 is mainly formed by transmembrane helix (TM) III, TM V, TM VI, and TM VII. Most of the residues contributing to ligand binding are positionally conserved comparing with other hTAS2Rs. By comparing the final conformations obtained by extensive MD simulations, we identified the changes in the transmembrane helices and the intra- and extracellular loops, which were expected to initiate the activation of the receptor. The intracellular loop II (ICL2) and TM III were found to play prominent roles in the process of activation. We proposed that a set of interactions between the aromatic Phe115 in the middle of ICL2 and three residues (Tyr103, Lys106, and Val107) at the cytoplasmic end of TM III may serve as a conformational switch of hTAS2R1 activation. All of the residues involved in the switch are highly conserved among T2Rs. This indicates that the control switch we proposed may be universal in T2Rs. Besides, our results also suggest that the formation of a short helical segment in ICL2 may be necessary for the activation of hTAS2R1.     

Estrogen-induced upregulation and 3′-UTR shortening of CDC6

3′-Untranslated region (UTR) shortening of mRNAs via alternative polyadenylation (APA) has important ramifications for gene expression. By using proximal APA sites and switching to shorter 3′-UTRs, proliferating cells avoid miRNA-mediated repression. Such APA and 3′-UTR shortening events may explain the basis of some of the proto-oncogene activation cases observed in cancer cells. In this study, we investigated whether 17 β-estradiol (E2), a potent proliferation signal, induces APA and 3′-UTR shortening to activate proto-oncogenes in estrogen receptor positive (ER+) breast cancers. Our initial probe based screen of independent expression arrays suggested upregulation and 3′-UTR shortening of an essential regulator of DNA replication, CDC6 (cell division cycle 6), upon E2 treatment. We further confirmed the E2- and ER-dependent upregulation and 3′UTR shortening of CDC6, which lead to increased CDC6 protein levels and higher BrdU incorporation. Consequently, miRNA binding predictions and dual luciferase assays suggested that 3′-UTR shortening of CDC6 was a mechanism to avoid 3′-UTR-dependent negative regulations. Hence, we demonstrated CDC6 APA induction by the proliferative effect of E2 in ER+ cells and provided new insights into the complex regulation of APA. E2-induced APA is likely to be an important but previously overlooked mechanism of E2-responsive gene expression.