Marked increase in PROP taste responsiveness following oral supplementation with selected salivary proteins or their related free amino acids

The genetic predisposition to taste 6-n-propylthiouracil (PROP) varies among individuals and is associated with salivary levels of Ps-1 and II-2 peptides, belonging to the basic proline-rich protein family (bPRP). We evaluated the role of these proteins and free amino acids that selectively interact with the PROP molecule, in modulating bitter taste responsiveness. Subjects were classified by their PROP taster status based on ratings of perceived taste intensity for PROP and NaCl solutions. Quantitative and qualitative determinations of Ps-1 and II-2 proteins in unstimulated saliva were performed by HPLC-ESI-MS analysis. Subjects rated PROP bitterness after supplementation with Ps-1 and II-2, and two amino acids (L-Arg and L-Lys) whose interaction with PROP was demonstrated by (1)H-NMR spectroscopy. ANOVA showed that salivary levels of II-2 and Ps-1 proteins were higher in unstimulated saliva of PROP super-tasters and medium tasters than in non-tasters. Supplementation of Ps-1 protein in individuals lacking it in saliva enhanced their PROP bitter taste responsiveness, and this effect was specific to the non-taster group.(1)H-NMR results showed that the interaction between PROP and L-Arg is stronger than that involving L-Lys, and taste experiments confirmed that oral supplementation with these two amino acids increased PROP bitterness intensity, more for L-Arg than for L-Lys. These data suggest that Ps-1 protein facilitates PROP bitter taste perception and identifies a role for free L-Arg and L-Lys in PROP tasting

The gustin (CA6) gene polymorphism, rs2274333 (A/G), as a mechanistic link between PROP tasting and fungiform taste papilla density and maintenance

Taste sensitivity to PROP varies greatly among individuals and is associated with polymorphisms in the bitter receptor gene TAS2R38, and with differences in fungiform papilla density on the anterior tongue surface. Recently we showed that the PROP non-taster phenotype is strongly associated with the G variant of polymorphism rs2274333 (A/G) of the gene that controls the salivary trophic factor, gustin. The aims of this study were 1) to investigate the role of gustin gene polymorphism rs2274333 (A/G), in PROP sensitivity and fungiform papilla density and morphology, and 2) to investigate the effect of this gustin gene polymorphism on cell proliferation and metabolic activity. Sixty-four subjects were genotyped for both genes by PCR techniques, their PROP sensitivity was assessed by scaling and threshold methods, and their fungiform papilla density, diameter and morphology were determined. In vitro experiments examined cell proliferation and metabolic activity, following treatment with saliva of individuals with and without the gustin gene mutation, and with isolated protein, in the two iso-forms. Gustin and TAS2R38 genotypes were associated with PROP threshold (p=0.0001 and p=0.0042), but bitterness intensity was mostly determined by TAS2R38 genotypes (p<0.000001). Fungiform papillae densities were associated with both genotypes (p<0.014) (with a stronger effect for gustin; p=0.0006), but papilla morphology was a function of gustin alone (p<0.0012). Treatment of isolated cells with saliva from individuals with the AA form of gustin or direct application of the active iso-form of gustin protein increased cell proliferation and metabolic activity (p<0.0135). These novel findings suggest that the rs2274333 polymorphism of the gustin gene affects PROP sensitivity by acting on fungiform papilla development and maintenance, and could provide the first mechanistic explanation for why PROP super-tasters are more responsive to a broad range of oral stimul

3D Structure Prediction of TAS2R38 Bitter Receptors Bound to Agonists Phenylthiocarbamide (PTC) and 6-n-Propylthiouracil (PROP)

The G protein-coupled receptor (GPCR) TAS2R38 is a bitter taste receptor that can respond to bitter compounds such as phenylthiocarbamide (PTC) and 6-n-propylthiouracil (PROP). This receptor was chosen because its four haplotypes (based on three residue site polymorphism) hTAS2R38_PAV, hTAS2R38_AVI, hTAS2R38_AAI, and hTAS2R38_PVV are known to have dramatically different responses to PTC and PROP. We aimed to identify the protein–ligand interaction features that determine whether the bitter taste signal from this receptor is sent to the cortex. To do this we predicted the 3D structures of the TAS2R38 bitter taste receptor using our new BiHelix and SuperBiHelix Monte Carlo methods (No experimental determinations of the 3D structure have been reported for any taste receptors.). We find that residue 262 (2nd position in the polymorphism) is involved in the interhelical hydrogen bond network stabilizing the GPCR structure in tasters (hTAS2R38_PAV, hTAS2R38_AAI, and hTAS2R38_PVV), while it is not in the nontaster (hTAS2R38_AVI). This suggests that the hydrogen bond interactions between TM3 and TM6 or between TM5 and TM6 may play a role in activating this GPCR. To further validate these structures, we used the DarwinDock method to predict the binding sites and 3D structures for PTC and PROP bound to hTAS2R38_PAV, hTAS2R38_AVI, hTAS2R38_AAI, and hTAS2R38_PVV, respectively. Our results show that PTC and PROP can form H-bonds with the backbone of residue 262 in the tasters (hTAS2R38_PAV, hTAS2R38_AAI, and hTAS2R38_PVV) but not in the nontaster (hTAS2R38_AVI). Thus it appears that the hydrogen bond interaction between TM3 and TM6 may activate the receptor to pass the ligand binding signal to intracellular processes and that the H-bond between agonists and residue 262 in tasters is involved in the bitter tasting. This is in agreement with experimental observations, providing validation of the predicted ligand-protein complexes and also a potential activation mechanism for the TAS2R38 receptor

Knowledge Based Membrane Protein Structure Prediction: From X-Ray Crystallography to Bioinformatics and Back to Molecular Biology

Integral membrane proteins play a key role in detecting and conveying outside signals into cells, allowing them to interact and respond to their environment in a specific manner. They form principal nodes in several signaling pathways and attract large interest in therapeutic interventions as the majority of drug targets are associated to the cell's membrane. The original human genome sequence project estimated 20% of the total gene count of 31,778 genes to code for membrane proteins[1]. Thus membrane proteins constitute a very large set of yet-to be-characterized proteins mediating all the relevant life-related functions both in prokaryotes and eukaryotes. Estimates are suggesting that in whole genomes the content of this protein type may vary from 10% to 40% of the whole proteome, depending on the organism. As of today, this may change on time, while the rough amount of protein sequences is ~ 6,000,000 (in the Non Redundant data base [http://www.ncbi.nlm.nih.gov/]), the sequences annotated as \u201cmembrane protein\u201d are just 45,281 in Swiss-Prot (http://expasy.org/sprot/ where the annotation is manually curated), and the solved atomic structures of membrane proteins are about 350 in the Protein Data Bank [http://www.rcsb.org/pdb/]. This is a very small number considering that we may consider a rough average of 30% of membrane proteins per genome (as derived from sequence similarity search) and end up with an approximate number of about 2,000,000 membrane proteins in the data bases. We can then easily evaluate that less than ~0.6% of membrane proteins are annotated and that ~0.001% of all the membrane protein sequences are known with atomic resolution , giving the idea of an enormous gap that should be filled in order to fully characterize the functioning of membrane proteins. The main reason behind these small numbers is that membrane proteins are very difficult to study as they are inserted into lipid bilayers surrounding the cell and its subcompartments, and expose to the polar outer and inner environments portions of different sizes. When isolated from membranes, membrane proteins are generally less stable than globular ones. It is therefore difficult to purify them in the native, functional form, and more difficult to crystallize them. Thus, crystallization of this type of proteins is yet a very difficult process, given the fact that they expose two different chemico-physical surfaces to the environment: water- and lipid-like. Still, in the last few years, and after great improvements in the techniques underlying X-ray crystallography, several new membrane proteins were solved in different activation states, offering to the entire scientific community a fundamental contribution to the characterization of astonishing mechanisms of signal transduction. Although the improvements in the technologies allowed the determination of several new structures in the last few years, the gap between the known membrane proteins and those with solved structures is still enormous. Thus, a deep combination of X-ray crystallography techniques, computational biology techniques and molecular biology validating experiments, is the key to face the challenge of bridging the gap existing between membrane proteins with and those without known structures. This and other issues may be resolved in the post-genomic era by taking advantage of the all the theoretical and experimental efforts aiming at developing tools based on our present knowledge that are capable of extracting selected structural/functional features from known sequences/structures and of computing the likelihood of their presence in never-seen before sequences/structures. Indeed, some of state-of-the-art tools, are based in the seminal idea that proteins are products of evolution and that their sequences contain millions of years of evolutionary information waiting to be extracted

Sensitivity to chemical stimuli plays a fundamental role in the food preferences. Examples in the evolutionary scale: 1. Role of the walking leg chemoreceptors in the red swamp crayfish Procambarus Clarkii 2. PROP bitter taste sensitivity and its nutritional implications in Humans

In this thesis, we studied two examples of the sensitivity to chemical stimuli and its role in the food preferences in two models of the evolutionary scale. The red swamp crayfish Procambarus clarkii (Girard, 1852) (Crustacea: Decapoda) is an invasive species of freshwater habitats that has spread worldwide. In crayfish, like in other decapod crustaceans, reception of chemical cues occurs by way of peripheral chemoreceptors grouped within sensory hairs and typically located on the cuticle of cephalothoracic appendages. Antennules and pereopods (walking legs), in particular, have been reported to be olfactory organs involved in a number of behavioral responses, such as, sex recognition and localization of food sources in the environment. By way of extracellular nerve recordings coupled with behavioral bioassays, we investigated the sensitivity spectra of the walking leg chemoreceptors in the crayfish P. clarkii in response to different compounds of feeding significance and related to its omnivorous habits. Our results confirmed a marked sensitivity of the legs to trehalose, cellobiose, sucrose, maltose, glycine and leucine. Some sensitivity to glucose, fructose, asparagine (all food indicators) and taurocholic acid was also found, the sugar-sensitive chemoreceptor units resulting as broadly tuned to the carbohydrates. Responses were highly phasic to trehalose (hemolymph sugar in the body fluid of many invertebrates), phasic to glycine and leucine and phasic-tonic to the other compounds. This suggests that chemoreceptor phasicity is an additional property for better discrimination of the protein components in the diet from other stimuli. The behavioral bioassays excluded, at least under confined experimental conditions, any involvement of antennules in the detection of food-related compounds, thus emphasizing the role of the crayfish legs as the main short-distance, broad-spectrum sensors for feeding. Such information may be valuable for the identification of key chemicals aimed at the future development of strategies for crayfish population control programs. Taste sensitivity varies greatly in humans, influencing eating behavior and therefore may play a role in body composition. PROP bitter taste sensitivity is the most studied example of the individual variability of taste sensitivity. Some studies show that PROP bitter taste sensitivity may be correlated with sensitivity to other oral stimuli, food preferences and BMI, while other studies did not confirm this association. It is known that PROP phenotype is associated with variant in bitter taste receptors TAS2R38 and with density of fungiform papillae on tongue surface. Although most of PROP phenotypic variations are explained by the allelic diversity of the bitter receptor TAS2R38, they cannot explain the PROP taster status-related differences above all that in the perception to different oral stimuli. The aim of this study was identify and characterize other factors that may contribute to differences in the genetic predisposition to taste PROP and identify confounding variables which may explain the controversial data in the literature about the relationship between PROP taste sensitivity and BMI. 1) We investigated the possible relationship between PROP bitter taste responsiveness and salivary proteins by using HPLC-ESI-MS on saliva sample before and after PROP taste stimulation. 2) We evaluated the role of proteins and free amino acids in modulating bitter taste responsiveness. Subjects rated PROP bitterness after supplementation of two salivary proteins (Ps-1 and II-2), and the free form of constituent amino acids of the two proteins sequences (L-Arg and L-Lys) whose interaction with PROP was demonstrated by 1H-NMR spectroscopy. 3) We investigate the role of polymorphism rs2274333 (A/G) in the gene that codify for the salivary trofic factor gustin protein, in PROP sensitivity and fungiform papilla density and morphology and in vitro we investigate the effect of this gustin gene polymorphism on cell proliferation and metabolic activity, following treatment with saliva of individuals with and without the gustin gene mutation, and with isolated protein, in the two iso-forms. 4) We investigated whether the endocannabinoid system, which modulates hunger/satiety and energy balance, plays a role in modulating eating behaviour influenced by a sensitivity to PROP which could explain the controversial data in literature. In particular we determined the plasma profile of the endocannabinoids 2-arachidonoylglycerol (2-AG), anandamide (AEA) and congeners in normal-weight PROP super-tasters and non-tasters, also we assessed the cognitive eating behavior disorder by the Three-Factor Eating Questionnaire. The results showed that: 1) Basal levels of II-2 and Ps-1 proteins, belonging to the basic proline-rich protein (bPRPs) family, were significantly higher in PROP super-taster than in non-taster unstimulated saliva, and PROP stimulation elicited a rapid increase in the levels of these same proteins only in PROP super-taster saliva. 2) Supplementation of Ps-1 protein in individuals lacking it in saliva enhanced their PROP bitter responsiveness. 1H-NMR results showed that the interaction between PROP and L-Arg is stonger than that involving L-Lys, and taste experiments confirmed that oral supplementation with L-Arg increase more PROP bitterness intensity than L-Lys. 3) Gustin and TAS2R38 genotypes were associated with PROP threshold, while bitterness intensity was mostly determined by TAS2R38 genotypes. Fungiform papillae densities were associated with both genotypes (with a stronger effect for gustin), but papilla morphology was a function of gustin alone. In vitro experiment, the treatment of isolated cells with saliva from individuals with AA form, and direct application of the active iso-form of gustin protein, increased cell proliferation and metabolic activity. 4) The disinhibition score of non-taster was higher than those of super-tasters. In addition, we found that the concentration of endocannabinoid AEA (anandamide) and 2-AG (2-arachidonoylglycerol) was lower in the plasma of non taster compared with super-tasters subjects. In conclusion, among the factors contributing to individual differences of PROP sensitivity, in addition to the TAS2R38 variants with its different affinity for the stimulus, we found: 1-2) the specific salivary proteins of bPRP family (Ps-1) and L-Arg that could be involved in twist and turn of the PROP molecule, thus facilitating its binding with the receptor. 3) A gustin gene polymorphism that, by modulating the protein activity, controls the growth and maintenance of taste buds and 4) the higher disinhibition behaviour in non-tasters may be compensated in part, in normal-weight subjects, by the decrease of peripheral endocannabinoids to downregulate the hunger-energy intake circuitry

Dose-dependent effects of L-Arginine on PROP bitterness intensity and latency and characteristics of the chemical interaction between PROP and L-Arginine

Genetic variation in the ability to taste the bitterness of 6-n-propylthiouracil (PROP) is a complex trait that has been used to predict food preferences and eating habits. PROP tasting is primarily controlled by polymorphisms in the TAS2R38 gene. However, a variety of factors are known to modify the phenotype. Principle among them is the salivary protein Ps-1 belonging to the basic proline-rich protein family (bPRP). Recently, we showed that oral supplementation with Ps-1 as well as its related free amino acids (L-Arg and L-Lys) enhances PROP bitterness perception, especially for PROP non-tasters who have low salivary levels of Ps-1. Here, we show that salivary L-Arg levels are higher in PROP super-tasters compared to medium tasters and non-tasters, and that oral supplementation with free L-Arg enhances PROP bitterness intensity as well as reduces bitterness latency in a dose-dependent manner, particularly in individuals with low salivary levels of both free L-Arg and Ps-1 protein. Supplementation with L-Arg also enhanced the bitterness of caffeine. We also used 1H-NMR spectroscopy and quantum-mechanical calculations carried out by Density Functional Theory (DFT) to characterize the chemical interaction between free L-Arg and the PROP molecule. Results showed that the -NH2 terminal group of the L-ArgH+ side chain interacts with the carbonyl or thiocarbonyl groups of PROP by forming two hydrogen bonds with the resulting charged adduct. The formation of this PROP•ArgH+ hydrogen-bonded adduct could enhance bitterness intensity by increasing the solubility of PROP in saliva and its availability to receptor sites. Our data suggest that L-Arg could act as a 'carrier' of various bitter molecules in saliva

Dose-Dependent Effects of L-Arginine on PROP Bitterness Intensity and Latency and Characteristics of the Chemical Interaction between PROP and L-Arginine

Genetic variation in the ability to taste the bitterness of 6-n-propylthiouracil (PROP) is a complex trait that has been used to predict food preferences and eating habits. PROP tasting is primarily controlled by polymorphisms in the TAS2R38 gene. However, a variety of factors are known to modify the phenotype. Principle among them is the salivary protein Ps-1 belonging to the basic proline-rich protein family (bPRP). Recently, we showed that oral supplementation with Ps-1 as well as its related free amino acids (L-Arg and L-Lys) enhances PROP bitterness perception, especially for PROP non-tasters who have low salivary levels of Ps-1. Here, we show that salivary L-Arg levels are higher in PROP super-tasters compared to medium tasters and non-tasters, and that oral supplementation with free L-Arg enhances PROP bitterness intensity as well as reduces bitterness latency in a dose-dependent manner, particularly in individuals with low salivary levels of both free L-Arg and Ps-1 protein. Supplementation with L-Arg also enhanced the bitterness of caffeine. We also used 1H-NMR spectroscopy and quantum-mechanical calculations carried out by Density Functional Theory (DFT) to characterize the chemical interaction between free L-Arg and the PROP molecule. Results showed that the -NH2 terminal group of the L-ArgH+ side chain interacts with the carbonyl or thiocarbonyl groups of PROP by forming two hydrogen bonds with the resulting charged adduct. The formation of this PROP\u2022ArgH+ hydrogen-bonded adduct could enhance bitterness intensity by increasing the solubility of PROP in saliva and its availability to receptor sites. Our data suggest that L-Arg could act as a 'carrier' of various bitter molecules in saliva

Genetic Study of Phenylthiocarbamide (PTC) Taste Sensitivity In Population of The Osing in Kemiren Village-Banyuwangi

The ability to taste phenylthiocarbamide (PTC), is autosomal trait inherited in a simple Mendelian recessive pattern. The frequency of Taster and non-Taster allele is varies in different populations. The purpose of the research is to investigate the prevalence, gene frequency and genotype frequency of taster (T) and non taster (ts of Osing population in Kemiren-Banyuwangi. PTC serial dilution method was used to assess the PTC Taster and non-Taster phenotypes. The Hardy–Weinberg method was used to determine allele frequencies. The total of samples were 227 people, male were 117 and female were 110 with age range of 15–30 years were randomly selected. The result showed that the Osing population as Taster were 210 (92,52%) and non Taster were 17samples (7,48%) . The allele frecuency of Taster (T) was 0,73 and non Taster (t) was 0,27 respectively. The genotype frequency of dominant Taster (TT) was 0,54, heterozygosity Taster (Tt) was 0,39, and genotype of non Taster (tt) was 0,07