RESUMEN
Many animal and plant species synthesize toxic compounds as deterrent; thus, detection of these compounds is of vital importance to avoid their ingestion. Often, such compounds are recognized by taste 2 receptors that mediate bitter taste in humans. Until now, bitter taste receptors have only been found in bony vertebrates, where they occur as a large family already in coelacanth, a "living fossil" and the earliest-diverging extant lobe-finned fish. Here, we have revisited the evolutionary origin of taste 2 receptors (T2Rs) making use of a multitude of recently available cartilaginous fish genomes. We have identified a singular T2R in 12 cartilaginous fish species (9 sharks, 1 sawfish, and 2 skates), which represents a sister clade to all bony fish T2Rs. We have examined its ligands for two shark species, a catshark and a bamboo shark. The ligand repertoire of bamboo shark represents a subset of that of the catshark, with roughly similar thresholds. Amarogentin, one of the most bitter natural substances for humans, also elicited the highest signal amplitudes with both shark receptors. Other subsets of ligands are shared with basal bony fish T2Rs indicating an astonishing degree of functional conservation over nearly 500 mya of separate evolution. Both shark receptors respond to endogenous steroids as well as xenobiotic compounds, whereas separate receptors exist for xenobiotics both in early- and late-derived bony vertebrates (coelacanth, zebrafish, and human), consistent with the shark T2R reflecting the original ligand repertoire of the ancestral bitter taste receptor at the evolutionary origin of this family.
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Tiburones , Gusto , Animales , Humanos , Gusto/fisiología , Receptores Acoplados a Proteínas G/genética , Percepción del Gusto/genética , Ligandos , Pez Cebra , Tiburones/genéticaRESUMEN
The vertebrate sense of taste allows rapid assessment of the nutritional quality and potential presence of harmful substances prior to ingestion. Among the five basic taste qualities, salty, sour, sweet, umami, and bitter, bitterness is associated with the presence of putative toxic substances and elicits rejection behaviors in a wide range of animals including humans. However, not all bitter substances are harmful, some are thought to be health-beneficial and nutritious. Among those compound classes that elicit a bitter taste although being non-toxic and partly even essential for humans are bitter peptides and L-amino acids. Using functional heterologous expression assays, we observed that the 5 dominant human bitter taste receptors responsive to bitter peptides and amino acids are activated by bile acids, which are notorious for their extreme bitterness. We further demonstrate that the cross-reactivity of bitter taste receptors for these two different compound classes is evolutionary conserved and can be traced back to the amphibian lineage. Moreover, we show that the cross-detection by some receptors relies on "structural mimicry" between the very bitter peptide L-Trp-Trp-Trp and bile acids, whereas other receptors exhibit a phylogenetic conservation of this trait. As some bile acid-sensitive bitter taste receptor genes fulfill dual-roles in gustatory and non-gustatory systems, we suggest that the phylogenetic conservation of the rather surprising cross-detection of the two substance classes could rely on a gene-sharing-like mechanism in which the non-gustatory function accounts for the bitter taste response to amino acids and peptides.
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Ácidos y Sales Biliares , Péptidos , Receptores Acoplados a Proteínas G , Gusto , Ácidos y Sales Biliares/metabolismo , Humanos , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , Animales , Gusto/fisiología , Péptidos/metabolismo , Filogenia , Células HEK293 , Aminoácidos/metabolismo , Membrana Celular/metabolismoRESUMEN
Arabica coffee contains the bitter-tasting diterpene glycoside mozambioside, which degrades during coffee roasting, leading to yet unknown structurally related degradation products with possibly similar bitter-receptor-activating properties. The study aimed at the generation, isolation, and structure elucidation of individual pyrolysis products of mozambioside and characterization of bitter receptor activation by in vitro analysis in HEK 293T-Gα16gust44 cells. The new compounds 17-O-ß-d-glucosyl-11-hydroxycafestol-2-on, 11-O-ß-d-glucosyl-16-desoxycafestol-2-on, 11-O-ß-d-glucosyl-(S)-16-desoxy-17-oxocafestol-2-on, 11-O-ß-d-glucosyl-15,16-dehydrocafestol-2-on, and 11-O-ß-d-glucosyl-(R)-16-desoxy-17-oxocafestol-2-on were isolated from pyrolyzed mozambioside by HPLC and identified by NMR and UHPLC-ToF-MS. Roasting products 11-O-ß-d-glucosyl-(S)-16-desoxy-17-oxocafestol-2-on, 11-O-ß-d-glucosyl-15,16-dehydrocafestol-2-on, and 11-O-ß-d-glucosyl-(R)-16-desoxy-17-oxocafestol-2-on had lower bitter receptor activation thresholds compared to mozambioside. Molecular docking simulations revealed the binding modes of the compounds 11-O-ß-d-glucosyl-15,16-dehydrocafestol-2-on and 11-O-ß-d-glucosyl-(R)-16-desoxy-17-oxocafestol-2-on and their aglycone 11-hydroxycafestol-2-on in the two cognate receptors TAS2R43 and TAS2R46. The newly discovered roasting products 17-O-ß-d-glucosyl-11-hydroxycafestol-2-on, 11-O-ß-d-glucosyl-(S)-16-desoxy-17-oxocafestol-2-on, 11-O-ß-d-glucosyl-15,16-dehydrocafestol-2-on, and 11-O-ß-d-glucosyl-(R)-16-desoxy-17-oxocafestol-2-on were detected in authentic roast coffee brew by UHPLC-ToF-MS and could contribute to coffee's bitter taste impression.
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Glicósidos , Gusto , Simulación del Acoplamiento Molecular , Cromatografía Líquida de Alta Presión , Espectroscopía de Resonancia MagnéticaRESUMEN
SCOPE: To avoid ingestion of potentially harmful substances, humans are equipped with about 25 bitter taste receptor genes (TAS2R) expressed in oral taste cells. Humans exhibit considerable variance in their bitter tasting abilities, which are associated with genetic polymorphisms in bitter taste receptor genes. One of these variant receptor genes, TAS2R2, is initially believed to represent a pseudogene. However, TAS2R2 exists in a putative functional variant within some populations and can therefore be considered as an additional functional bitter taste receptor. METHODS AND RESULTS: To learn more about the function of the experimentally neglected TAS2R2, a functional screening with 122 bitter compounds is performed. The study observes responses with eight of the 122 bitter substances and identifies the substance phenylbutazone as a unique activator of TAS2R2 among the family of TAS2Rs, thus filling one more gap in the array of cognate bitter substances. CONCLUSIONS: The comprehensive characterization of the receptive range of TAS2R2 allows the classification into the group of TAS2Rs with a medium number of bitter agonists. The variability of bitter taste and its potential influences on food choice in some human populations may be even higher than assumed.
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Receptores Acoplados a Proteínas G , Gusto , Humanos , Gusto/genética , Receptores Acoplados a Proteínas G/genética , Percepción del Gusto/genéticaRESUMEN
Despite the important role of bitter taste for the rejection of potentially harmful food sources, birds have long been suspected to exhibit inferior bitter tasting abilities. Although more recent reports on the bitter recognition spectra of several bird species have cast doubt about the validity of this assumption, the bitter taste of avian species is still an understudied field. Previously, we reported the bitter activation profiles of three zebra finch receptors Tas2r5, -r6, and -r7, which represent orthologs of a single chicken bitter taste receptor, Tas2r1. In order to get a better understanding of the bitter tasting capabilities of zebra finches, we selected another Tas2r gene of this species that is similar to another chicken Tas2r. Using functional calcium mobilization experiments, we screened zebra finch Tas2r1 with 72 bitter compounds and observed responses for 7 substances. Interestingly, all but one of the newly identified bitter agonists were different from those previously identified for Tas2r5, -r6, and -r7 suggesting that the newly investigated receptor fills important gaps in the zebra finch bitter recognition profile. The most potent bitter agonist found in our study is cucurbitacin I, a highly toxic natural bitter substance. We conclude that zebra finch exhibits an exquisitely developed bitter taste with pronounced cucurbitacin I sensitivity suggesting a prominent ecological role of this compound for zebra finch.
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Flavor is perceived through the olfactory, taste, and trigeminal systems, mediated by designated GPCRs and channels. Signal integration occurs mainly in the brain, but some cross-reactivities occur at the receptor level. Here, we predict potential bitterness and taste receptors targets for thousands of odorants. BitterPredict and BitterIntense classifiers suggest that 3-9% of flavor and food odorants have bitter taste, but almost none are intensely bitter. About 14% of bitter molecules are expected to have an odor. Bitterness is more common for unpleasant smells such as fishy, amine, and ammoniacal, while non-bitter odorants often have pleasant smells. Experimental toxicity values suggest that fishy ammoniac smells are more toxic than pleasant smells, regardless of bitterness. TAS2R14 is predicted as the main bitter receptor for odorants, confirmed by in vitro profiling of 10 odorants. The activity of bitter odorants may have implications for physiology due to ectopic expression of taste and smell receptors.
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Neuronas Receptoras Olfatorias , Gusto , Humanos , Gusto/fisiología , Odorantes/análisis , Percepción del Gusto/fisiología , Olfato , Neuronas Receptoras Olfatorias/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
Taste perception is crucial for the critical evaluation of food constituents in human and other vertebrates. The five basic taste qualities salty, sour, sweet, umami (in humans mainly the taste of L-glutamic acid) and bitter provide important information on the energy content, the concentration of electrolytes and the presence of potentially harmful components in food items. Detection of the various taste stimuli is facilitated by specialized receptor proteins that are expressed in taste buds distributed on the tongue and the oral cavity. Whereas, salty and sour receptors represent ion channels, the receptors for sweet, umami and bitter belong to the G protein-coupled receptor superfamily. In particular, the G protein-coupled taste receptors have been located in a growing number of tissues outside the oral cavity, where they mediate important processes. This article will provide a brief introduction into the human taste perception, the corresponding receptive molecules and their signal transduction. Then, we will focus on taste receptors in the gastrointestinal tract, which participate in a variety of processes including the regulation of metabolic functions, hunger/satiety regulation as well as in digestion and pathogen defense reactions. These important non-gustatory functions suggest that complex selective forces have contributed to shape taste receptors during evolution.
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The composition of menus and the sequence of foodstuffs consumed during a meal underlies elaborate rules. However, the molecular foundations for the observed taste- and pleasure-raising effects of complex menus are obscure. The molecular identification and characterization of taste receptors can help to gain insight into the complex interrelationships of food items and beverages during meals. In our study, we quantified important bitter compounds in chicory and chicory-based surrogate coffee and used them to identify responsive bitter taste receptors. The two receptors, TAS2R43 and TAS2R46, are exquisitely sensitive to lactucin, lactucopicrin, and 11ß,13-dihydrolactucin. Sensory testing demonstrated a profound influence of the sequence of consumption of chicory, surrogate coffee, and roasted coffee on the perceived bitterness by human volunteers. These findings pave the way for a molecular understanding of some of the mixture effects underlying empirical meal compositions.
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Linseed oil is rich in unsaturated fatty acids, and its increased consumption could aid in health-promoting nutrition. However, rapid oxidation of linseed oil and concomitant development of bitterness impair consumers' acceptance. Previous research revealed that cyclolinopeptides, a group of cyclic peptides inherent to linseed oil, dominantly contribute to the observed bitterness. In the present study, fresh and stored linseed oil and flaxseed were analyzed for the presence of cyclolinopeptides using preparative high-performance liquid chromatography combined with mass spectrometry- and nuclear magnetic resonance-based identification and quantification. The purified compounds were tested for the activation of all 25 human bitter taste receptors of which only two responded exclusively to methionine-oxidized cyclolinopeptides. Of those, the methionine sulfoxide-containing cyclolinopeptide-4 elicited responses at relevant concentrations. We conclude that this compound is the main determinant of linseed oil's bitterness and propose strategies to reduce the development of bitterness.
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Lino , Aceite de Linaza , Anciano , Cromatografía Líquida de Alta Presión/métodos , Lino/química , Humanos , Aceite de Linaza/química , Péptidos Cíclicos/química , GustoRESUMEN
Bitterness is an aversive cue elicited by thousands of chemically diverse compounds. Bitter taste may prevent consumption of foods and jeopardize drug compliance. The G protein-coupled receptors for bitter taste, TAS2Rs, have species-dependent number of subtypes and varying expression levels in extraoral tissues. Molecular recognition by TAS2R subtypes is physiologically important, and presents a challenging case study for ligand-receptor matchmaking. Inspired by hybrid recommendation systems, we developed a new set of similarity features, and created the BitterMatch algorithm that predicts associations of ligands to receptors with ~ 80% precision at ~ 50% recall. Associations for several compounds were tested in-vitro, resulting in 80% precision and 42% recall. The encouraging performance was achieved by including receptor properties and integrating experimentally determined ligand-receptor associations with chemical ligand-to-ligand similarities.BitterMatch can predict off-targets for bitter drugs, identify novel ligands and guide flavor design. The novel features capture information regarding the molecules and their receptors, which could inform various chemoinformatic tasks. Inclusion of neighbor-informed similarities improves as experimental data mounts, and provides a generalizable framework for molecule-biotarget matching.
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Coffee is one of the most consumed hot beverages worldwide and is highly regarded because of its stimulating effect despite having a pronounced bitterness. Even though numerous bitter ingredients have been identified, the detailed molecular basis for coffee's bitterness is not well understood except for caffeine, which activates five human bitter taste receptors. We elucidated the contribution of other bitter coffee constituents in addition to caffeine with functional calcium imaging experiments using mammalian cells expressing the cDNAs of human bitter taste receptors, sensory experiments, and in silico modeling approaches. We identified two human bitter taste receptors, TAS2R43 and TAS2R46, that responded to the bitter substance mozambioside with much higher sensitivity than to caffeine. Further, the structurally related bitter substances bengalensol, cafestol, and kahweol also activated the same pair of bitter taste receptors much more potently than the prototypical coffee bitter substance caffeine. However, for kahweol, a potent but weak activator of TAS2R43 and TAS2R46, we observed an inhibitory effect when simultaneously applied together with mozambioside to TAS2R43 expressing cells. Molecular modeling experiments showed overlapping binding sites in the receptor's ligand binding cavity that suggest that the partial agonist kahweol might be useful to reduce the overall bitterness of coffee-containing beverages. Taken together, we found that the bitterness of coffee is determined by a complex interaction of multiple bitter compounds with several human bitter taste receptors.
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Coffea/metabolismo , Aromatizantes/metabolismo , Cafeína/química , Cafeína/metabolismo , Coffea/química , Aromatizantes/química , Humanos , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Semillas/química , Semillas/metabolismo , GustoRESUMEN
Targeted analysis of Coffea arabica and Coffea canephora green coffees (total sample size n = 57) confirmed 2- O-ß-d-glucopyranosyl-carboxyatractyligenin (6) as the quantitatively dominating carboxyatractyligenin derivative. Its abundance in Arabicas (2425 ± 549 nmol/g, n = 48) exceeded that in Robustas (34 ± 12 nmol/g, n = 9) roughly by a factor of 70. Coffee processing involving heat (e.g., steam treatment and decaffeination) reduced concentrations of 6 and increased those of the decarboxylated derivative. The bioavailability of compound 6 in Caenorhabditis elegans was demonstrated by ultraperformance liquid chromatography-tandem mass spectrometry analysis of extracts prepared from nematode cultures incubated in a liquid medium containing 6. A toxicity assay performed to assess the impact of 6 in vivo showed a 20-fold higher median lethal dose (LD50 = 11.7 ± 1.2 mM) concentration compared to that of the known phytotoxic adenine-nucleotide transporters inhibitor carboxyatractyloside (2, LD50 = 0.61 ± 0.05 mM), whereas 1 mM 6 and 0.1 mM 2 were sufficient to decrease the survival of wild type C. elegans, already 10-20-fold lower doses reduced reproduction. Because the insulin/insulin-like growth factors signaling cascade (IIS) is a key regulator of life span and stress resistance, the impact of compound 6 on the survival of long-living daf-2 C. elegans was tested. As the susceptibility of these nematodes to 6 was as high as that in wild type, an impact on central metabolic processes independent of IIS was suggested. Analysis of the in vivo adenosine triphosphate (ATP) content of adult C. elegans revealed no changes after 1 and 24 h, but a 50% reduction after treatment with 1 mM 6 during the entire postembryonic development. These data speak for a developmental-stage-dependent modulation of the ATP pool by 6.
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Atractilósido/análogos & derivados , Caenorhabditis elegans/efectos de los fármacos , Coffea/química , Preparaciones de Plantas/farmacología , Adenosina Trifosfato/metabolismo , Animales , Atractilósido/farmacocinética , Atractilósido/farmacología , Disponibilidad Biológica , Caenorhabditis elegans/genética , Caenorhabditis elegans/crecimiento & desarrollo , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Coffea/toxicidad , Café/química , Femenino , Insulina/genética , Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/metabolismo , Dosificación Letal Mediana , MasculinoRESUMEN
To validate the suitability of synephrine, known to be a highly abundant alkaloid in oranges, as a dietary biomarker for orange consumption, a highly sensitive and robust stable isotope dilution analysis (SIDA) as well as an ECHO method, using the analyte itself as a pseudointernal standard injected into the analysis run to provide an "echo peak" of the analyte, was developed to quantitate synephrine by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in citrus juices and human urine before and after the ingestion of orange juice. A citrus juice screening revealed high synephrine concentrations of 150-420 nmol/mL in orange (n = 11) and tangerine (n = 2) juices, whereas 20-100 times lower levels were found in juice from grapefruit (n = 14), lemon (n = 5), pomelo (n = 2), and lime (n = 4). Application of the SIDA to quantitate synephrine in sulfatase/glucuronidase-treated urine samples (n = 10) after orange juice consumption showed an increase of synephrine from trace levels (0.1 ± 0.1 nmol/mL) in the 2-day washout phase to a maximum concentration of 8.9 (±5.5) nmol/mL found 4 h after ingestion of orange juice. Whereas proline betaine was recently reported as a dietary biomarker indicating the ingestion of any citrus product and Chinese artichoke, synephrine can be used a reliable additional biomarker with high specificity for orange and tangerine.
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Biomarcadores/orina , Cromatografía Líquida de Alta Presión/métodos , Citrus sinensis/metabolismo , Sinefrina/orina , Espectrometría de Masas en Tándem/métodos , Adulto , Femenino , Frutas/metabolismo , Humanos , Masculino , Adulto JovenRESUMEN
Proline betaine has been proposed as a candidate dietary biomarker for citrus intake. To validate its suitability as a dietary biomarker and to gain insight into the range of this per-methylated amino acid in foods and beverages, a quick and accurate stable isotope dilution assay was developed for quantitative high-throughput HILIC-MS/MS screening of proline betaine in foods and urine after solvent-mediated matrix precipitation. Quantitative analysis of a variety of foods confirmed substantial amounts of proline betaine in citrus juices (140-1100 mg/L) and revealed high abundance in tubers of the vegetable Stachys affinis, also known as Chinese artichocke (â¼700 mg/kg). Seafood including clams, shrimp, and lobster contained limited amounts (1-95 mg/kg), whereas only traces were detected in fish, cuttlefish, fresh meat, dairy products, fresh vegetable (<3 mg/kg), coffee, tea, beer, and wine (<7 mg/L). The human excretion profiles of proline betaine in urine were comparable when common portions of orange juice or fried Stachys tubers were consumed. Neither mussels nor beer provided enough proline betaine to detect significant differences between morning urine samples collected before and after consumption. As Stachys is a rather rare vegetable and not part of peoples' daily diet, the data reported here will help to monitor the subject's compliance in future nutritional human studies on citrus products or the exclusion of citrus products in the wash-out phase of an intervention study. Moreover, proline betaine measurement can contribute to the establishment of a toolbox of valid dietary biomarkers reflecting wider aspects of diet to assess metabolic profiles as measures of dietary exposure and indicators of dietary patterns, dietary changes, or effectiveness of dietary interventions.
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Betaína/orina , Bebidas/análisis , Citrus/metabolismo , Frutas/metabolismo , Prolina/orina , Adulto , Betaína/metabolismo , Biomarcadores/metabolismo , Biomarcadores/orina , Femenino , Ensayos Analíticos de Alto Rendimiento , Humanos , Masculino , Prolina/metabolismo , Espectrometría de Masas en Tándem , Adulto JovenRESUMEN
Capsules, powders and tablets containing raw coffee extract are advertised to the consumer as antioxidant rich dietary supplements as part of a healthy diet. We isolated carboxyatractyligenin (4), 2-O-ß-d-glucopyranosyl carboxyatractyligenin (6) and 3'-O-ß-d-glucopyranosyl-2'-O-isovaleryl-2ß-(2-desoxy-carboxyatractyligenin)-ß-d-glucopyranoside (8) from green coffee and found strong inhibitory effects on phosphorylating respiration in isolated mitochondria similar to the effects of the known phytotoxin carboxyatractyloside. LC-MS/MS analysis of commercial green coffee based dietary supplements revealed the occurrence of carboxyatractyligenin, 3'-O-ß-d-glucopyranosyl-2'-O-isovaleryl-2ß-(2-desoxy-carboxyatractyligenin)-ß-d-glucopyranoside, and 2-O-ß-d-glucopyranosyl carboxyatractyligenin in concentrations up to 4.0, 5.7, and 41.6µmol/g, respectively. These data might help to gain first insight into potential physiological side-effects of green coffee containing dietary supplement.