Niemann–Pick C1-Like 1 (NPC1L1) mediates cholesterol absorption, and ezetimibe is a potent NPC1L1 inhibitor applicable for medication of hypercholesterolemia. Epidemiological studies demonstrated that consumption of polyphenols correlates with a decreased risk for atherosclerosis due to their antioxidant effect. This activity can hardly be attributable to the antioxidant activity only, and we hypothesized that polyphenols inhibit intestinal transport of cholesterol. We elucidated the kinetic parameters of intestinal cholesterol absorption, screened several polyphenols for their ability to specifically inhibit intestinal cholesterol absorption, and determined the inhibitory effects of selected flavonoids in vitro and in vivo. The concentration-dependent uptake of cholesterol by Caco-2 cells obeyed a monophasic saturation process. This indicates the involvement of an active-passive transport, i.e., NPC1L1. Parameters of cholesterol uptake by Caco-2 cells were as follows: Jmax, Kt, and Kd were 6.89±2.96 19.03±11.58 µM, and 0.11±0.02 pmol/min/mg protein, respectively. Luteolin and quercetin inhibited cholesterol absorption by Caco-2 cells and human embryonic kidney 293T cells expressing NPC1L1. When preincubated Caco-2 cells with luteolin and quercetin before the assay, cholesterol uptake significantly decreased. The inhibitory effects of these flavonoids were maintained for up to 120 min. The level of inhibition and irreversible effects were similar to that of ezetimibe. Serum cholesterol levels significantly decreased more in rats fed both cholesterol and luteolin (or quercetin), than in those observed in the cholesterol feeding group. As quercetin induced a significant decrease in the levels of NPC1L1 mRNA in Caco-2 cells, the in vivo inhibitory effect may be due to the expression of NPC1L1. These results suggest that luteolin and quercetin reduce high blood cholesterol levels by specifically inhibiting intestinal cholesterol absorption mediated by NPC1L1.
To elucidate the effect of the polyphenols contained in alcoholic beverages on the metabolic stress induced by ethanol consumption, four groups of mice were fed for five weeks on Lieber's diet with or without ethanol, with ethanol plus ellagic acid, and with ethanol plus trans-resveratrol. Alcoholic fatty liver was observed in the group fed the ethanol diet but not in those fed the ethanol plus polyphenol diets. Liver transcriptome analysis revealed that the addition of the polyphenols suppressed the expression of the genes related to cell stress that were up-regulated by ethanol alone. Conversely, the polyphenols up-regulated the genes involved in bile acid synthesis, unsaturated fatty acid elongation, and tetrahydrofolate synthesis that were down-regulated by ethanol alone. Because parts of these genes were known to be regulated by the constitutive androstane receptor (CAR), we performed the same experiment in the CAR-deficient mice. As a result, fatty liver was observed not only in the ethanol group but also with the ethanol plus polyphenol groups. In addition, there was no segregation of the gene expression profiles among these groups. These results provide a molecular basis for the prevention of alcohol-induced stress by the polyphenols in alcoholic beverages.
Background: T1R1/T1R3 exhibits species-dependent differences in ligand specificity.
Results: The ligand specificity is dependent on a combination of amino acid selectivity at the orthosteric site and receptor activity modulation at the non-orthosteric site.
Conclusion: The molecular mechanism underlying the amino acid recognition of T1R1/T1R3 has been elucidated.
Significance: This study provides new insights into the molecular mechanisms of the l-Glu-specific response in human T1R1/T1R3.
Umami taste perception in mammals is mediated by a heteromeric complex of two G-protein-coupled receptors, T1R1 and T1R3. T1R1/T1R3 exhibits species-dependent differences in ligand specificity; human T1R1/T1R3 specifically responds to l-Glu, whereas mouse T1R1/T1R3 responds more strongly to other l-amino acids than to l-Glu. The mechanism underlying this species difference remains unknown. In this study we analyzed chimeric human-mouse receptors and point mutants of T1R1/T1R3 and identified 12 key residues that modulate amino acid recognition in the human- and mouse-type responses in the extracellular Venus flytrap domain of T1R1. Molecular modeling revealed that the residues critical for human-type acidic amino acid recognition were located at the orthosteric ligand binding site. In contrast, all of the key residues for the mouse-type broad response were located at regions outside of both the orthosteric ligand binding site and the allosteric binding site for inosine-5′-monophosphate (IMP), a known natural umami taste enhancer. Site-directed mutagenesis demonstrated that the newly identified key residues for the mouse-type responses modulated receptor activity in a manner distinct from that of the allosteric modulation via IMP. Analyses of multiple point mutants suggested that the combination of two distinct determinants, amino acid selectivity at the orthosteric site and receptor activity modulation at the non-orthosteric sites, may mediate the ligand specificity of T1R1/T1R3. This hypothesis was supported by the results of studies using nonhuman primate T1R1 receptors. A complex molecular mechanism involving changes in the properties of both the orthosteric and non-orthosteric sites of T1R1 underlies the determination of ligand specificity in mammalian T1R1/T1R3.
Amino Acid; G Protein-coupled Receptors (GPCR); Glutamate Receptors Metabotropic; Mutagenesis in Vitro; Receptor Structure-Function; Taste Receptor; Umami
In mammals, bitter taste is mediated by TAS2R genes, which belong to the large family of seven transmembrane G protein-coupled receptors. Because TAS2Rs are directly involved in the interaction between mammals and their dietary sources, it is likely that these genes evolved to reflect species-specific diets during mammalian evolution. Here, we investigated the sensitivities of TAS2R16s of various primates by using a cultured cell expression system, and found that the sensitivity of each primate species varied according to the ligand. Especially, the sensitivity of TAS2R16 of Japanese macaques to salicin was much lower than that of human TAS2R16, which was supported by behavioural tests. These results suggest the possibility that bitter-taste sensitivities evolved independently by replacing specific amino acid residues of TAS2Rs in different primate species to adapt to food items they use.
old world monkey; bitter taste receptor; chimpanzee
Brazilian green propolis is a popular health supplement because of its various biological properties. The ethanol extract of Brazilian green propolis (EEBP) is characteristic for its herb-like smell and unique pungent taste. However, the ingredients responsible for its pungency have not yet been identified. This study provides the first evidence that artepillin C is the main pungent ingredient in EEBP and that it potently activates human transient receptor potential ankyrin 1 (TRPA1) channels. EEBP was fractionated using column chromatography with a step gradient elution of an ethanol-water solution, and the fractions having the pungent taste were determined by sensory tests. HPLC analysis revealed that the pungent fraction was composed primarily of artepillin C, a prenylated derivative of cinnamic acid. Artepillin C was also identified as the pungent compound of EEBP by organoleptic examiners. Furthermore, the effects of artepillin C and other cinnamic acids found in EEBP on TRPA1 channels were examined by calcium imaging and plate reader-based assays in human TRPA1-expressing cells to investigate the molecular mechanisms underlying their pungent tastes. Artepillin C and baccharin activated the TRPA1 channel strongly, whereas drupanin caused a slight activation and p-coumaric acid showed no activation. Because the EC50 values of artepillin C, baccharin, and allyl isothiocyanate were 1.8 µM, 15.5 µM, and 6.2 µM, respectively, artepillin C was more potent than the typical TRPA1 agonist allyl isothiocyanate. These findings strongly indicate that artepillin C is the main pungent ingredient in EEBP and stimulates a pungent taste by activating TRPA1 channels.
TRPA1 and TRPV1 are members of the TRP superfamily of structurally related, nonselective cation channels. TRPA1 and TRPV1 are often co-expressed in sensory neurons and play an important role in somatosense such as cold, pain, and irritants. The first leaves of Kalopanax pictus Nakai (Araliaceae) have long been used as a culinary ingredient in Korea because of their unique chemesthetic flavor. In this study, we observed the intracellular Ca2+ response to cultured cells expressing human TRPA1 (hTRPA1) and human TRPV1 (hTRPV1) by Ca2+ imaging analysis to investigate the ability of the first leaves of K. pictus to activate the hTRPA1 and hTRPV1. An 80% ethanol extract of K. pictus (KPEx) increased intracellular Ca2+ influx in a response time- and concentration-dependent manner via either hTRPA1 or hTRPV1. KPEx-induced response to hTRPA1 was markedly attenuated by ruthenium red, a general blocker of TRP channels, and HC-030031, a specific antagonist of TRPA1. In addition, the intracellular Ca2+ influx attained with KPEx to hTRPV1 was mostly blocked by ruthenium red, and capsazepine, a specific antagonist of TRPV1. These results indicate that KPEx selectively activates both hTRPA1 and hTRPV1, which may provide evidence that the first leaves of K. pictus primarily activate TRPA1 and TRPV1 to induce their unique chemesthetic sense.
Kalopanax pictus; TRPA1; TRPV1; Ca2+ imaging; Chemesthesis
In this study, the glucansucrase from the dental caries pathogen S. mutans was purified and crystallized by the hanging-drop vapour-diffusion method using ammonium sulfate as a precipitant.
Glucansucrases encoded by Streptococcus mutans play essential roles in the synthesis of sticky dental plaques. Based on amino-acid sequence similarity, glucansucrases are classified as members of glycoside hydrolase family 70 (GH 70). Data on the crystal structure of GH 70 glucansucrases have yet to be reported. Here, the GH 70 glucansucrase GTF-SI from S. mutans was overexpressed in Escherichia coli strain BL21 (DE3), purified to homogeneity and crystallized using the hanging-drop vapour-diffusion method. Orthorhombic GTF-SI crystals belonging to space group P21212 were obtained. A diffraction data set was collected to 2.1 Å resolution.
glucansucrase; dental caries; Streptococcus mutans
One of the most distinctive features of human sweet taste perception is its broad tuning to chemically diverse compounds ranging from low-molecular-weight sweeteners to sweet-tasting proteins. Many reports suggest that the human sweet taste receptor (hT1R2–hT1R3), a heteromeric complex composed of T1R2 and T1R3 subunits belonging to the class C G protein–coupled receptor family, has multiple binding sites for these sweeteners. However, it remains unclear how the same receptor recognizes such diverse structures. Here we aim to characterize the modes of binding between hT1R2–hT1R3 and low-molecular-weight sweet compounds by functional analysis of a series of site-directed mutants and by molecular modeling–based docking simulation at the binding pocket formed on the large extracellular amino-terminal domain (ATD) of hT1R2. We successfully determined the amino acid residues responsible for binding to sweeteners in the cleft of hT1R2 ATD. Our results suggest that individual ligands have sets of specific residues for binding in correspondence with the chemical structures and other residues responsible for interacting with multiple ligands.
Neoculin occurring in the tropical fruit of Curculigo latifolia is currently the only protein that possesses both a sweet taste and a taste-modifying activity of converting sourness into sweetness. Structurally, this protein is a heterodimer consisting of a neoculin acidic subunit (NAS) and a neoculin basic subunit (NBS). Recently, we found that a neoculin variant in which all five histidine residues are replaced with alanine elicits intense sweetness at both neutral and acidic pH but has no taste-modifying activity. To identify the critical histidine residue(s) responsible for this activity, we produced a series of His-to-Ala neoculin variants and evaluated their sweetness levels using cell-based calcium imaging and a human sensory test. Our results suggest that NBS His11 functions as a primary pH sensor for neoculin to elicit taste modification. Neoculin variants with substitutions other than His-to-Ala were further analyzed to clarify the role of the NBS position 11 in the taste-modifying activity. We found that the aromatic character of the amino acid side chain is necessary to elicit the pH-dependent sweetness. Interestingly, since the His-to-Tyr variant is a novel taste-modifying protein with alternative pH sensitivity, the position 11 in NBS can be critical to modulate the pH-dependent activity of neoculin. These findings are important for understanding the pH-sensitive functional changes in proteinaceous ligands in general and the interaction of taste receptor–taste substance in particular.
The constitutive androstane receptor (CAR) is known as a xeno-sensor that regulates genes involved in xenobiotic excretion and energy metabolism. This study tested a variety of polyphenols for their ability to modulate CAR activity. HepG2 cells were transfected with a CAR expression plasmid and a reporter plasmid containing the human CYP2B6 regulatory region and then treated with flavonoids, catechins and other bioactive polyphenols. Luciferase assays revealed that baicalein (5, 6, 7-OH flavone) was a potent activator of both human and mouse CAR. Catechin gallates also activated human and mouse CAR. Wild-type and CAR knockout mice were treated with baicalein and chrysin (5, 7-OH flavone), and their liver mRNA was analyzed by real-time PCR. A significant increase in cyp2b10 mRNA content was observed only in wild-type mice fed chrysin. These results suggest that dietary flavonoids regulate CAR activity and thereby accelerate both detoxification and energy metabolism.
flavonoid; catechin; chrysin; constitutive androstane receptor; pregnane X receptor; cyp2b10; detoxification; energy metabolism
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.
Calcium Imaging; G-protein-coupled Receptors (GPCR); Membrane Proteins; Receptor Structure-Function; Signal Transduction; Bitter; Taste Receptor; Binding Site; Molecular Dynamics; Molecular Modeling
Autosomal dominant optic atrophy (DOA) is the most common form of hereditary optic neuropathy. DOA presents in the first decade of life and manifests as progressive vision loss. In DOA retinal ganglion cells and the optic nerve degenerate by an unknown mechanism. The gene mutated in DOA, Optic Atrophy Type 1 (OPA1), encodes a dynamin-related GTPase implicated in mitochondrial fusion and maintenance of the mitochondrial network and genome. Here, we determine which cell types in the normal retina and the optic nerve express OPA1. In the normal rat retina, OPA1 is expressed in the ganglion cell layer as well as in the outer plexiform layer, the inner nuclear layer, and the inner plexiform layer. In the ganglion cell layer, OPA1 is expressed predominantly in retinal ganglion cells. By contrast, OPA1 protein is low or undetectable in astrocytes and oligodendrocytes of the optic nerve. Additionally, OPA1 protein is present in axonal mitochondria. Last, OPA1 expression is present in mitochondria of processes and cell bodies of purified retinal ganglion cells and of the RGC-5 cell line. Thus, OPA1 is predominantly expressed in retinal ganglion cells of the normal rat retina and axons of the optic nerve. These findings may explain the selective vulnerability of retinal ganglion cells to OPA1 loss of function.
immunohistochemistry; retinal ganglion cells; mitochondria; dominant optic atrophy; dynamin-related GTPase
Neoculin (NCL), a protein with sweetness approximately 500-fold that of sugar, can be utilized as a nonglycemic sweetener. It also has taste-modifying activity to convert sourness to sweetness. NCL is a heterodimer composed of an N-glycosylated acidic subunit (NAS) and a basic subunit (NBS), which are conjugated by disulfide bonds. For the production of recombinant NCL (rNCL) by Aspergillus oryzae, α-amylase with a KEX2 cleavage site, -K-R-, was fused upstream of each of NAS and NBS and the resulting fusion proteins were simultaneously expressed. For accurate and efficient cleavage of the fusion construct by KEX2-like protease, a triglycine motif was inserted after the KEX2 cleavage site. As NBS showed lower production efficiency than did NAS, a larger amount of the NBS expression plasmid than of NAS expression plasmid was introduced during cotransformation, resulting in successful production of rNCL in the culture medium. Moreover, to obtain a higher production yield of rNCL, the active form of hacA cDNA encoding a transcription factor that induces an unfolded protein response was cloned and expressed constitutively. This resulted in a 1.5-fold increase in the level of rNCL production (2.0 mg/liter). rNCL was purified by chromatography, and its NAS was found to be N-glycosylated as expected. The original sweetness and taste-modifying activity of rNCL were comparable to those of native NCL when confirmed by calcium imaging with human embryonic kidney cells expressing the human sweet taste receptor and by sensory tests.
Polycystic kidney disease (PKD) 2L1 protein is a member of the transient receptor potential (TRP) ion channel family. In circumvallate and foliate papillae, PKD2L1 is coexpressed with PKD1L3. PKD2L1 and PKD1L3 interact through their transmembrane domain and the resulting heteromer PKD1L3/PKD2L1 owns a unique channel property called ‘off-responses’ to acid stimulation, although PKD2L1 does not own this property by itself. To define the pharmacological properties of the PKD1L3/PKD2L1 channel, we developed a new method to effectively evaluate channel activity using human embryonic kidney 293T cells in which the channel was heterologously expressed. This method was applied to screen substances that potentially regulate it. We found that capsaicin and its analogs, which are TRPV1 agonists, inhibited the response to acid stimuli and that the capsaicin inhibition was reversible with an IC50 of 32.5 μm. Capsaicin and its analogs are thus useful tools for physiological analysis of PKD1L3/PKD2L1 function.
Nucleotide sequence data are available in the GenBank database under the accession numbers hTRPA1, BC148423 and hTRPV3, BC104866.
acid; calcium imaging; capsaicin; PKD1L3/PKD2L1; transient receptor potential channel