Identifying the interactions between natural, non-caloric sweeteners and the human sweet receptor by molecular docking.

Natural sweeteners, such as stevia and thaumatin, exert their sweet taste by specifically binding to sweet taste receptors. However, the molecular basis of their sweetening power remains to be ascertained. In the present study, we built a comparative model of the hT1R2 and hT1R3 subunits in order to characterize their interactions with natural, non-caloric sweeteners - from glycosylated terpenoids to sweet proteins - at the molecular level. The binding free energy between hT1R2-hT1R3 and sweeteners of different families shows a strong correlation with their sweetness intensity for both, small sweeteners (r = -0.89) and sweet proteins (r = -0.97). The correlation is further improved and generalized throughout all families of sweeteners evaluated, when EC50 values are used instead of relative intensities (r = -0.91). Altogether, these results contribute to a better understanding of the sweetness perception of these sweeteners, and promote the use of docking for better prediction of resulting sweetness.

Docking and Molecular Dynamics of Steviol Glycoside-Human Bitter Receptor Interactions.

Stevia is one of the sweeteners with the greatest consumer demand because of its natural origin and minimal calorie content. Steviol glycosides (SG) are the main active compounds present in the leaves of Stevia rebaudiana and are responsible for its sweetness. However, recent in vitro studies in HEK 293 cells revealed that SG specifically activate the hT2R4 and hT2R14 bitter taste receptors, triggering this mouth feel. The objective of this study was to characterize the interaction of SG with these two receptors at the molecular level. The results showed that SG have only one site for orthosteric binding to these receptors. The binding free energy (ΔGbinding) between the receptor and SG was negatively correlated with SG bitterness intensity, for both hT2R4 (r = -0.95) and hT2R14 (r = -0.89). We also determined, by steered molecular dynamics simulations, that the force required to extract stevioside from the receptors was greater than that required for rebaudioside A, in accordance with the ΔG values obtained by molecular docking. Finally, we identified the loop responsible for the activation by SG of both receptors. As a whole, these results contribute to a better understanding of the resulting off-flavor perception of these natural sweeteners in foods and beverages, allowing for better prediction, and control, of the resulting bitterness.

Correlation of T2R38 taste phenotype and in vitro biofilm formation from nonpolypoid chronic rhinosinusitis patients.

BACKGROUND: Sinonasal biofilms have been demonstrated in specimens collected from chronic rhinosinusitis (CRS) patients. Mounting evidence suggests that biofilms contribute to therapeutically recalcitrant CRS. Recently, the bitter taste receptor T2R38 has been implicated in the regulation of the sinonasal mucosal innate immune response. TAS2R38 gene polymorphisms affect receptor functionality and contribute to variations seen in sinonasal innate defense as well as taste perception reflected in gustatory sensitivity to the bitter compound phenylthiocarbamide (PTC). In a population of CRS patients with active infection or inflammation, we sought to determine if a correlation between T2R38 phenotype and in vitro biofilm formation existed. METHODS: Endoscopically guided sinonasal swabs were obtained prospectively from CRS (±polyp) patients with evidence of persistent inflammation or mucopurulence. In vitro biofilm formation was assessed with a modified Calgary Biofilm Detection Assay. Patients' phenotypic (functional) expression of the bitter taste receptor T2R38 was evaluated with a taste test including the compound PTC. Linear regression was used to determine the level of significance between mean in vitro biofilm formation levels and mean PTC taste test intensity ratings across CRS patients. RESULTS: Sinonasal swabs were obtained from 59 patients, with 42 of the 59 samples demonstrating in vitro biofilm formation. Analysis revealed an inverse linear association between in vitro biofilm formation and PTC taste intensity ratings (p = 0.019) for all patients. This association was exclusively driven by nonpolypoid CRS patients (p = 0.0026). CONCLUSION: In vitro biofilm formation from sinonasal clinical isolates is inversely correlated with PTC taste sensitivity in nonpolypoid CRS patients.

T2R38 genotype is correlated with sinonasal quality of life in homozygous ΔF508 cystic fibrosis patients.

BACKGROUND: Chronic rhinosinusitis (CRS) is very prevalent in the cystic fibrosis (CF) patient population, and leads to high morbidity and markedly decreased quality of life (QOL). Identification of genetic markers that contribute to CRS symptoms in these patients can allow for risk stratification and tailoring of medical and surgical treatments. T2R38 is a bitter taste receptor expressed in the sinonasal tract, and nonfunctional alleles of this receptor have been implicated in treatment-refractory CRS in non-CF patients. The purpose of this study is to investigate the significance of T2R38 genotype in the variability of sinonasal QOL and CRS disease severity in a sample of CF patients. METHODS: ΔF508 homozygous CF patients were recruited from the University of Pennsylvania Cystic Fibrosis Center and were genotyped for the TAS2R38 locus. To assess sinonasal symptom severity, a 22-item Sino-Nasal Outcome Test (SNOT-22) was collected from each patient. Additional demographic and medical history data was obtained at the time of patient enrollment. RESULTS: A total of 49 ΔF508 homozygous CF patients aged 18 to 32 years were included in the final SNOT-22 score analysis. Individuals with 2 functional T2R38 alleles (PAV/PAV) had significantly lower SNOT-22 scores (n = 49, p < 0.05). On further breakdown of SNOT-22 subcategories, rhinologic symptoms specifically were less severe in PAV/PAV patients than patients with other genotypes (n = 47, p < 0.05). CONCLUSION: Our investigation indicates that T2R38 genotype correlates both with SNOT-22 scores and rhinologic-specific QOL in ΔF508 homozygous CF patients.

Phenylthiocarbamide taste sensitivity is associated with sinonasal symptoms in healthy adults.

BACKGROUND: The bitter taste receptor T2R38, expressed in the tongue and nasal epithelium, has been shown to trigger sinonasal innate immunity contributing to the prevention of gram-negative upper airway bacterial infections. Common polymorphisms of the T2R38 gene, correlating with bitter taste sensitivity to phenylthiocarbamide (PTC), have been linked to differences in sinonasal innate immune response, with specific genotypes significantly more common in medically recalcitrant chronic rhinosinusitis patients. The purpose of this study was to examine this association between T2R38 function and sinonasal infection or symptoms in a healthy population. METHODS: A survey of the frequency of sinus infections, as well as other nasal symptoms such as colds, allergies, and overall nasal quality of life (nQOL), was administered to healthy adult participants. nQOL was measured using a 0 to 3 scale of worsening symptoms. A PTC compound taste strip was administered with T2R38 taste sensitivity classified as extremely, somewhat, or not sensitive. RESULTS: Among 217 participants (55% female, 70% Caucasian, 42% age 21 to 25 years), 30% did not detect bitterness (nontasters), 34% were moderate tasters, and 36% were "supertasters," experiencing a strong, unpalatable bitterness. Supertasters were associated with less frequent sinus infections (p = 0.04), and PTC sensitivity was predictive of nasal symptoms: Supertasters had the best nQOL scores, followed by moderate tasters and nontasters (means: 0.65, 0.81, 1.00, respectively; p = 0.014 for trend). There were no significant associations with other variables. CONCLUSION: This study provides evidence that T2R38 functionality in the tongue correlates with nasal symptoms in healthy individuals.

Seleção de aptâmeros que se ligam ao receptor humano para o gosto doce / Screening for aptamers that bind to the human sweet taste receptor (hT1R2/hT1R3)

Foi demonstrado que o gosto doce é transduzido por receptores acoplados a proteína G classe III (GPCRs), T1R2 e T1R3. Essas proteínas exibem longas extremidades amino-terminais que formam um domínio de ligação globular extracelular. Elas são expressas em células associadas ao gosto (células epiteliais que constituem os botões gustativos nas papilas gustativas), que respondem a moléculas associadas ao gosto doce. Quando T1R2 e T1R3 são co-expressas em células heterólogas, elas respondem, como heterômeros, a uma série de açúcares, alguns D-aminoácidos, edulcorantes artificiais e proteínas doces. Foi também demonstrado que o receptor humano T1R2/T1R3 para o gosto doce apresenta múltiplos sítios de ligação. Para melhor compreender a estrutura desse receptor e responder à pergunta de como um único quimiorreceptor pode ser responsivo a uma variedade de ligantes, foi utilizada a abordagem denominada evolução sistemática de ligantes por enriquecimento exponencial (SELEX) para isolar, a partir de uma biblioteca combinatória de oligonucleotídeos, aptâmeros de RNA resistentes a nuclease que se ligam ao receptor humano para o gosto doce com alta afinidade. Após um enriquecimento de doze ciclos do pool original de RNA contendo em torno de 1013 sequências diferentes (contra preparações de membrana de células HEK293T que expressam hT1R2/hT1R3) e outros ciclos de contrasseleção negativa (para eliminar moléculas de RNA que se ligam de forma inespecífica à membrana de nitrocelulose e a outras proteínas diferentes do alvo, ou seja, proteínas de membrana de células HEK293T selvagem), realizou-se a transcrição reversa do RNA seguida de amplificação por PCR e sequenciamento. Aptâmeros do ciclo 12 com sequências consenso foram selecionados, e a ligação de alguns deles com hT1R2/hT1R3 foi então avaliada. Cinco desses aptâmeros mostram claramente uma maior afinidade por células HEK293T que expressam hT1R2/hT1R3. Como segunda parte desta tese, estudamos outro receptor, denominado CD36, que, como o receptor T1R2/T1R3, é expresso na língua. Estudos indicam que ele age como receptor gustativo de gordura. Neste trabalho, verificamos que essa proteína é expressa em uma subpopulação de neurônios olfatórios presentes no epitélio olfatório, indicando que ela pode ter também uma função olfatória, ainda não caracterizada

It has been shown that sweet taste is transduced by the Class III G Protein-Coupled Receptors (GPCRs) T1R2 and T1R3, which show long N-termini that form a globular extracellular ligand-binding domain. These receptors are expressed in the taste cells (epithelial cells that constitute the taste buds in taste papillae) that respond to sweet tastants, and when T1R2 and T1R3 are coexpressed in heterologous cells, they respond, as heteromers, to a series of sugars, some D-amino acids, artificial sweeteners and sweet proteins. It has also been demonstrated that the sweet taste receptor has multiple binding sites. In order to better understand the structure of this receptor and answer the question of how a single chemoreceptor can respond to a variety of ligands, we used the combinatorial oligonucleotide library screening approach, denominated Systematic Evolution of Ligands by Exponential Enrichment (SELEX), to isolate nuclease-resistant RNA aptamers that bind to the human sweet taste receptor with high affinity. Following a twelve round enrichment of the previous random RNA pool containing around 1013 different sequences (against membrane preparations of hT1R2/hT1R3-expressing HEK293T cells) and negative counterselection cycles (to eliminate RNA molecules that bind nonspecifically to the nitrocellulose membrane and to proteins other than the target, that is, HEK293T cells membrane proteins), the RNA was reverse-transcribed for DNA sequencing. Aptamers from cycle 12 with consensus sequences were selected, and the binding of some of them to the human sweet taste receptor was then evaluated. Five out of the aptamers clearly show greater affinity for hT1R2/hT1R3-expressing HEK293T cells than for hT1R2/hT1R3-non-expressing HEK293T cells. In this thesis we have also analyzed another receptor, denominated CD36, which is also expressed in the tongue. Studies indicate that it acts as a receptor for fat. In this work, we found that CD36 is expressed in a subset of the olfactory neurons localized in the olfactory epithelium, indicating that it may also have an as yet uncharacterized olfactory function