Modeling the structure of the transmembrane domain of T1R3, a subunit of the sweet taste receptor, with neohesperidin dihydrochalcone using molecular dynamics simulation.

Neohesperidin dihydrochalcone (NHDC) is a sweetener, which interacts with the transmembrane domain (TMD) of the T1R3 subunit of the human sweet taste receptor. Although NHDC and a sweet taste inhibitor lactisole share similar structural motifs, they have opposite effects on the receptor. This study involved the creation of an NHDC-docked model of T1R3 TMD through mutational analyses followed by in silico simulations. When certain NHDC derivatives were docked to the model, His7345.44 was demonstrated to play a crucial role in activating T1R3 TMD. The NHDC-docked model was then compared with a lactisole-docked inactive form, several residues were characterized as important for the recognition of NHDC; however, most of them were distinct from those of lactisole. Residues such as His6413.33 and Gln7947.38 were found to be oriented differently. This study provides useful information that will facilitate the design of sweeteners and inhibitors that interact with T1R3 TMD.

A single residue confers selective loss of sugar sensing in wrynecks.

Sensory receptors evolve, and changes to their response profiles can directly impact sensory perception and affect diverse behaviors, from mate choice to foraging decisions.1-3 Although receptor sensitivities can be highly contingent on changes occurring early in a lineage's evolutionary history,4 subsequent shifts in a species' behavior and ecology may exert selective pressure to modify and even reverse sensory receptor capabilities.5-7 Neither the extent to which sensory reversion occurs nor the mechanisms underlying such shifts is well understood. Using receptor profiling and behavioral tests, we uncover both an early gain and an unexpected subsequent loss of sugar sensing in woodpeckers, a primarily insectivorous family of landbirds.8,9 Our analyses show that, similar to hummingbirds10 and songbirds,4 the ancestors of woodpeckers repurposed their T1R1-T1R3 savory receptor to detect sugars. Importantly, whereas woodpeckers seem to have broadly retained this ability, our experiments demonstrate that wrynecks (an enigmatic ant-eating group sister to all other woodpeckers) selectively lost sugar sensing through a novel mechanism involving a single amino acid change in the T1R3 transmembrane domain. The identification of this molecular microswitch responsible for a sensory shift in taste receptors provides an example of the molecular basis of a sensory reversion in vertebrates and offers novel insights into structure-function relationships during sensory receptor evolution.

Synergism, Bifunctionality, and the Evolution of a Gradual Sensory Trade-off in Hummingbird Taste Receptors.

Sensory receptor evolution can imply trade-offs between ligands, but the extent to which such trade-offs occur and the underlying processes shaping their evolution is not well understood. For example, hummingbirds have repurposed their ancestral savory receptor (T1R1-T1R3) to detect sugars, but the impact of this sensory shift on amino acid perception is unclear. Here, we use functional and behavioral approaches to show that the hummingbird T1R1-T1R3 acts as a bifunctional receptor responsive to both sugars and amino acids. Our comparative analyses reveal substantial functional diversity across the hummingbird radiation and suggest an evolutionary timeline for T1R1-T1R3 retuning. Finally, we identify a novel form of synergism between sugars and amino acids in vertebrate taste receptors. This work uncovers an unexplored axis of sensory diversity, suggesting new ways in which nectar chemistry and pollinator preferences can coevolve.

Evaluation of Cell-Free Synthesized Human Channel Proteins for In Vitro Channel Research.

Despite channel proteins being important drug targets, studies on channel proteins remain limited, as the proteins are difficult to express and require correct complex formation within membranes. Although several in vitro synthesized recombinant channels have been reported, considering the vast diversity of the structures and functions of channel proteins, it remains unclear which classes of channels cell-free synthesis can be applied to. In this study, we synthesized 250 clones of human channels, including ion channel pore-forming subunits, gap junction proteins, porins, and regulatory subunits, using a wheat cell-free membrane protein production system, and evaluated their synthetic efficiency and function. Western blotting confirmed that 95% of the channels were successfully synthesized, including very large channels with molecular weights of over 200 kDa. A subset of 47 voltage-gated potassium ion channels was further analyzed using a planar lipid bilayer assay, out of which 80% displayed a voltage-dependent opening in the assay. We co-synthesized KCNB1 and KCNS3, a known heteromeric complex pair, and demonstrated that these channels interact on a liposome. These results indicate that cell-free protein synthesis provides a promising solution for channel studies to overcome the bottleneck of in vitro protein production.

A chalcone derivative suppresses TSLP induction in mice and human keratinocytes through binding to BET family proteins.

Although treatments for allergic diseases have improved, side effects and treatment resistance remain as challenges. New therapeutic drugs for allergic diseases are urgently required. Thymic stromal lymphopoietin (TSLP) is a cytokine target for prevention and treatment of allergic diseases. Since TSLP is produced from epithelial cells in allergic diseases, TSLP inhibitors may be new anti-allergic drugs. We previously identified a new inhibitor of TSLP production, named 16D10. However, its target of action remained unclarified. In this study, we found proteins binding to 16D10 from 24,000 human protein arrays by AlphaScreen-based high-throughput screening and identified bromodomain and extra-terminal (BET) family proteins as targets. We also clarified the detailed mode of interaction between 16D10 and a BET family protein using X-ray crystallography. Furthermore, we confirmed that inhibitors of BET family proteins suppressed TSLP induction and IL-33 and IL-36γ expression in both mouse and human keratinocyte cell lines. Taken together, our findings suggest that BET family proteins are involved in the suppression of TSLP production by 16D10. These proteins can contribute to the pathology of atopic dermatitis via TSLP regulation in keratinocytes and have potential as therapeutic targets in allergic diseases.

Ibuprofen inhibits oral NaCl response through transmembrane channel-like 4.

Nonsteroidal anti-inflammatory drugs, such as ibuprofen, are known to modify salty taste perception in humans. However, the underlying molecular mechanisms remain unknown. We investigated the inhibitory effect of ibuprofen on the NaCl stimulation of epithelium sodium channel (ENaC) and transmembrane channel-like 4 (TMC4), which are involved in salty taste detection. Although ibuprofen only minimally inhibited the response of the ENaC to NaCl, it significantly inhibited the TMC4 response to NaCl with an IC50 at 1.45 mM. These results suggest that ibuprofen interferes with detection of salty taste via inhibition of TMC4.

Evolution of the primate glutamate taste sensor from a nucleotide sensor.

Taste perception plays an essential role in food selection. Umami (savory) tastes are sensed by a taste receptor complex, T1R1/T1R3, that detects proteinogenic amino acids.1 High sensitivity to l-glutamate (l-Glu) is a characteristic of human T1R1/T1R3, but the T1R1/T1R3 of other vertebrates does not consistently show this l-Glu response.1,2 Here, we demonstrate that the l-Glu sensitivity of T1R1/T1R3 is a derived state that has evolved repeatedly in large primates that rely on leaves as protein sources, after their divergence from insectivorous ancestors. Receptor expression experiments show that common amino acid substitutions at ligand binding sites that render T1R1/T1R3 sensitive to l-Glu occur independently at least three times in primate evolution. Meanwhile T1R1/T1R3 senses 5'-ribonucleotides as opposed to l-Glu in several mammalian species, including insectivorous primates. Our chemical analysis reveal that l-Glu is one of the major free amino acids in primate diets and that insects, but not leaves, contain large amounts of free 5'-ribonucleotides. Altering the ligand-binding preference of T1R1/T1R3 from 5'-ribonucleotides to l-Glu might promote leaf consumption, overcoming bitter and aversive tastes. Altogether, our results provide insight into the foraging ecology of a diverse mammalian radiation and help reveal how evolution of sensory genes facilitates invasion of new ecological niches.

Early origin of sweet perception in the songbird radiation.

Early events in the evolutionary history of a clade can shape the sensory systems of descendant lineages. Although the avian ancestor may not have had a sweet receptor, the widespread incidence of nectar-feeding birds suggests multiple acquisitions of sugar detection. In this study, we identify a single early sensory shift of the umami receptor (the T1R1-T1R3 heterodimer) that conferred sweet-sensing abilities in songbirds, a large evolutionary radiation containing nearly half of all living birds. We demonstrate sugar responses across species with diverse diets, uncover critical sites underlying carbohydrate detection, and identify the molecular basis of sensory convergence between songbirds and nectar-specialist hummingbirds. This early shift shaped the sensory biology of an entire radiation, emphasizing the role of contingency and providing an example of the genetic basis of convergence in avian evolution.

CNKSR1 serves as a scaffold to activate an EGFR phosphatase via exclusive interaction with RhoB-GTP.

Epidermal growth factor receptor (EGFR) and human EGFR 2 (HER2) phosphorylation drives HER2-positive breast cancer cell proliferation. Enforced activation of phosphatases for those receptors could be a therapeutic option for HER2-positive breast cancers. Here, we report that degradation of an endosomal small GTPase, RhoB, by the ubiquitin ligase complex cullin-3 (CUL3)/KCTD10 is essential for both EGFR and HER2 phosphorylation in HER2-positive breast cancer cells. Using human protein arrays produced in a wheat cell-free protein synthesis system, RhoB-GTP, and protein tyrosine phosphatase receptor type H (PTPRH) were identified as interacting proteins of connector enhancer of kinase suppressor of Ras1 (CNKSR1). Mechanistically, constitutive degradation of RhoB, which is mediated by the CUL3/KCTD10 E3 complex, enabled CNKSR1 to interact with PTPRH at the plasma membrane resulting in inactivation of EGFR phosphatase activity. Depletion of CUL3 or KCTD10 led to the accumulation of RhoB-GTP at the plasma membrane followed by its interaction with CNKSR1, which released activated PTPRH from CNKSR1. This study suggests a mechanism of PTPRH activation through the exclusive binding of RhoB-GTP to CNKSR1.

A comprehensive interaction study provides a potential domain interaction network of human death domain superfamily proteins.

Human death domain superfamily proteins (DDSPs) play important roles in many signaling pathways involved in cell death and inflammation. Disruption or constitutive activation of these DDSP interactions due to inherited gene mutations is closely related to immunodeficiency and/or autoinflammatory diseases; however, responsible gene mutations have not been found in phenotypical diagnosis of these diseases. In this study, we comprehensively investigated the interactions of death-fold domains to explore the signaling network mediated by human DDSPs. We obtained 116 domains of DDSPs and conducted a domain-domain interaction assay of 13,924 reactions in duplicate using amplified luminescent proximity homogeneous assay. The data were mostly consistent with previously reported interactions. We also found new possible interactions, including an interaction between the caspase recruitment domain (CARD) of CARD10 and the tandem CARD-CARD domain of NOD2, which was confirmed by reciprocal co-immunoprecipitation. This study enables prediction of the interaction network of human DDSPs, sheds light on pathogenic mechanisms, and will facilitate identification of drug targets for treatment of immunodeficiency and autoinflammatory diseases.

Recent progress in the use of diaziridine-based sweetener derivatives to elucidate the chemoreception mechanism of the sweet taste receptor.

All sweeteners are recognized by the sweet taste receptor (T1R2-T1R3). The elucidation of the chemoreception mechanism of receptor-ligand interactions is an attractive topic for researchers. Molecular biology and computational biology techniques can reveal the proposed mechanisms for this topic. Other approaches, including chemical biology (bioorganic chemistry), have helped to identify mechanisms on the basis of molecular structure. In this mini-review, we have summarized the recent progress in the synthesis of sweetener derivatives, which includes the use of photoaffinity labeling of diazirine-based derivatives to elucidate the chemoreception of sweeteners.

Vibrational analysis of acetylcholine binding to the M2 receptor.

The M2 muscarinic acetylcholine receptor (M2R) is a prototypical G protein-coupled receptor (GPCR) that responds to acetylcholine (ACh) and mediates various cellular responses in the nervous system. We recently established Attenuated Total Reflection-Fourier Transform Infrared (ATR-FTIR) spectroscopy for ligand binding to M2R reconstituted in lipid membranes, paving the way to understand the mechanism in atomic detail. However, the obtained difference FTIR spectra upon ligand binding contained ligand, protein, lipid, and water signals, so a vibrational assignment was needed for a thorough understanding. In the present study, we compared difference FTIR spectra between unlabeled and 2-13C labeled ACh, and assigned the bands at 1741 and 1246 cm-1 as the C[double bond, length as m-dash]O and C-O stretches of ACh, respectively. The C[double bond, length as m-dash]O stretch of ACh in M2R is close to that in aqueous solution (1736 cm-1), and much lower in frequency than the free C[double bond, length as m-dash]O stretch (1778-1794 cm-1), indicating a strong hydrogen bond, which probably formed with N4046.52. We propose that a water molecule bridges ACh and N4046.52. The other ACh terminal is positively charged, and it interacts with negatively charged D1033.32. The present study revealed that D1033.32 is deprotonated (negatively charged) in both ACh-bound and free states, a suggested mechanism to stabilize the negative charge of D1033.32 in the free M2R.

Ibuprofen, a Nonsteroidal Anti-Inflammatory Drug, is a Potent Inhibitor of the Human Sweet Taste Receptor.

A sweet taste receptor is composed of heterodimeric G-protein-coupled receptors T1R2 and T1R3. Although there are many sweet tastants, only a few compounds have been reported as negative allosteric modulators (NAMs), such as lactisole, its structural derivative 2,4-DP, and gymnemic acid. In this study, candidates for NAMs of the sweet taste receptor were explored, focusing on the structural motif of lactisole. Ibuprofen, a nonsteroidal anti-inflammatory drug (NSAID), has an α-methylacetic acid moiety, and this structure is also shared by lactisole and 2,4-DP. When ibuprofen was applied together with 1 mM aspartame to the cells that stably expressed the sweet taste receptor, it inhibited the receptor activity in a dose-dependent manner. The IC50 value of ibuprofen against the human sweet taste receptor was calculated as approximately 12 µM, and it was almost equal to that of 2,4-DP, which is known as the most potent NAM for the receptor to date. On the other hand, when the inhibitory activities of other profens were examined, naproxen also showed relatively potent NAM activity against the receptor. The results from both mutant analysis for the transmembrane domain (TMD) of T1R3 and docking simulation strongly suggest that ibuprofen and naproxen interact with T1R3-TMD, similar to lactisole and 2,4-DP. However, although 2,4-DP and ibuprofen had almost the same inhibitory activities, these activities were acquired by filling different spaces of the ligand pocket of T1R3-TMD; this knowledge could lead to the rational design of a novel NAM against the sweet taste receptor.

Asymmetric Synthesis of Photophore-Containing Lactisole Derivatives to Elucidate Sweet Taste Receptors.

Lactisole, which has a 2-phenoxy propionic acid skeleton, is well-known as an inhibitor of sweet taste receptors. We recently revealed some of the structure-activity relationships of the aromatic ring and chiral center of lactisole. Photoaffinity labeling is one of the common chemical biology methods to elucidate the interaction between bioactive compounds and biomolecules. In this paper, the novel asymmetric synthesis of lactisole derivatives with common photophores (benzophenone, azide and trifluoromethyldiazirine) for photoaffinity labeling is described. The synthetic compounds are subjected to cell-based sweet taste receptors, and the substitution with trifluoromethyldiazirinyl photophore shows the highest affinity to the receptor of the synthesized compounds.

Structural insights into the differences among lactisole derivatives in inhibitory mechanisms against the human sweet taste receptor.

Lactisole, an inhibitor of the human sweet taste receptor, has a 2-phenoxypropionic acid skeleton and has been shown to interact with the transmembrane domain of the T1R3 subunit (T1R3-TMD) of the receptor. Another inhibitor, 2,4-DP, which shares the same molecular skeleton as lactisole, was confirmed to be approximately 10-fold more potent in its inhibitory activity than lactisole; however the structural basis of their inhibitory mechanisms against the receptor remains to be elucidated. Crystal structures of the TMD of metabotropic glutamate receptors, which along with T1Rs are categorized as class C G-protein coupled receptors, have recently been reported and made it possible to create an accurate structural model for T1R3-TMD. In this study, the detailed structural mechanism underlying sweet taste inhibition was characterized by comparing the action of lactisole on T1R3-TMD with that of 2,4-DP. We first performed a series of experiments using cultured cells expressing the sweet taste receptor with mutations and examined the interactions with these inhibitors. Based on the results, we next performed docking simulations and then applied molecular dynamics-based energy minimization. Our analyses clearly revealed that the (S)-isomers of both lactisole and 2,4-DP, interacted with the same seven residues in T1R3-TMD and that the inhibitory potencies of those inhibitors were mainly due to stabilizing interactions mediated via their carboxyl groups in the vertical dimension of the ligand pocket of T1R3-TMD. In addition, 2,4-DP engaged in a hydrophobic interaction mediated by its o-Cl group, and this interaction may be chiefly responsible for the higher inhibitory potency of 2,4-DP.

Ligand binding to human prostaglandin E receptor EP4 at the lipid-bilayer interface.

Prostaglandin E receptor EP4, a G-protein-coupled receptor, is involved in disorders such as cancer and autoimmune disease. Here, we report the crystal structure of human EP4 in complex with its antagonist ONO-AE3-208 and an inhibitory antibody at 3.2 Å resolution. The structure reveals that the extracellular surface is occluded by the extracellular loops and that the antagonist lies at the interface with the lipid bilayer, proximal to the highly conserved Arg316 residue in the seventh transmembrane domain. Functional and docking studies demonstrate that the natural agonist PGE2 binds in a similar manner. This structural information also provides insight into the ligand entry pathway from the membrane bilayer to the EP4 binding pocket. Furthermore, the structure reveals that the antibody allosterically affects the ligand binding of EP4. These results should facilitate the design of new therapeutic drugs targeting both orthosteric and allosteric sites in this receptor family.

Positive/Negative Allosteric Modulation Switching in an Umami Taste Receptor (T1R1/T1R3) by a Natural Flavor Compound, Methional.

Taste is a vital sensation for vertebrates, enabling the detection of nutritionally important substances or potential toxins. A heteromeric complex of two class C GPCRs, T1R1 and T1R3, was identified as the umami (savory) taste receptor. Amino acids and 5'-ribonucleotides are well known to be natural ligands for human T1R1/T1R3. In this study, we reveal that methional, which is a familiar flavor component in foods, is an allosteric modulator of T1R1/T1R3. Receptor expression experiments showed that methional served as a positive allosteric modulator (PAM) of human T1R1/T1R3 and functioned as a negative allosteric modulator (NAM) of mouse T1R1/T1R3. Although amino acids and 5'-ribonucleotides bound to the extracellular domain of T1R1, the use of interspecies chimeric receptors demonstrated that methional interacted with the transmembrane domain of T1R1. Site-directed mutagenesis and molecular modeling showed that methional could potentially bind at two distinct sites in the transmembrane domain of T1R1 and that the amino acid residues in the bottom of the allosteric pocket engendered the switch between the PAM and NAM modes, which could contribute to switching the binding position of methional. These results may be applicable for elucidating the molecular mechanisms underlying ligand recognition by other class C GPCRs.

Sensory biology. Evolution of sweet taste perception in hummingbirds by transformation of the ancestral umami receptor.

Sensory systems define an animal's capacity for perception and can evolve to promote survival in new environmental niches. We have uncovered a noncanonical mechanism for sweet taste perception that evolved in hummingbirds since their divergence from insectivorous swifts, their closest relatives. We observed the widespread absence in birds of an essential subunit (T1R2) of the only known vertebrate sweet receptor, raising questions about how specialized nectar feeders such as hummingbirds sense sugars. Receptor expression studies revealed that the ancestral umami receptor (the T1R1-T1R3 heterodimer) was repurposed in hummingbirds to function as a carbohydrate receptor. Furthermore, the molecular recognition properties of T1R1-T1R3 guided taste behavior in captive and wild hummingbirds. We propose that changing taste receptor function enabled hummingbirds to perceive and use nectar, facilitating the massive radiation of hummingbird species.

L-Theanine elicits umami taste via the T1R1 + T1R3 umami taste receptor.

L-Theanine is a unique amino acid present in green tea. It elicits umami taste and has a considerable effect on tea taste and quality. We investigated L-theanine activity on the T1R1 + T1R3 umami taste receptor. L-Theanine activated T1R1 + T1R3-expressing cells and showed a synergistic response with inosine 5'-monophosphate. The site-directed mutagenesis analysis revealed that L-theanine binds to L-amino acid binding site in the Venus flytrap domain of T1R1. This study shows that L-theanine elicits an umami taste via T1R1 + T1R3.