Discovery of natural TRPA1 activators through pharmacophore-based virtual screening and a biological assay.

Transient receptor potential cation channel subfamily A member 1 (TRPA1), a member of the transient receptor potential family, detects a wide range of environmental stimuli, such as low temperature, abnormal pH, and reactive irritants. TRPA1 is of great interest as a target protein in fields related to pharmaceuticals and foods. In this study, a library of natural products was explored to identify TRPA1 activators by pharmacophore screening of known TRPA1 agonists and biological assays for agonist activity. The study identified six natural compounds as novel TRPA1 agonists. The discovery of these compounds may prove useful in elucidating the TRPA1 activation mechanism.

Identification of a new class of non-electrophilic TRPA1 agonists by a structure-based virtual screening approach.

Recent work has gradually been clarifying the binding site of non-electrophilic agonists on the transient receptor potential A1 (TRPA1). This study searched for non-electrophilic TRPA1 agonists by means of in silico drug discovery techniques based on three-dimensional (3-D) protein structure. First, agonist-bound pocket structures were explored using an advanced molecular dynamics simulation starting from the cryo-electron microscopic structure of TRPA1, and several pocket structures suitable for virtual screening were extracted by structure evaluation using known non-electrophilic TRPA1 agonists. Next, 49 compounds were selected as new non-electrophilic agonist candidates from a library of natural products comprising 10,555 compounds by molecular docking toward these pocket structures. Measurement of the TRPA1 agonist activity of these compounds showed notable TRPA1 activation with three compounds (decanol, 2-ethyl-1-hexanol, phenethyl butanoate). Decanol and 2-ethyl-1-hexanol, which are categorized as fatty alcohols, in particular have a novel chemical scaffold for TRPA1 activation. The results of this study are expected to be of considerable use in understanding the molecular mechanism of TRPA1 recognition by non-electrophilic agonists.

Expression of Bitter Taste Receptors in the Intestinal Cells of Non-Human Primates.

(1) Background: Recent studies have investigated the expression of taste-related genes in the organs of various animals, including humans; however, data for additional taxa are needed to facilitate comparative analyses within and among species. (2) Methods: We investigated the expression of taste-related genes in the intestines of rhesus macaques, the non-human primates most commonly used in experimental models. (3) Results: Based on RNAseq and qRT-PCR, genes encoding bitter taste receptors and the G-protein gustducin were expressed in the gut of rhesus macaques. RNAscope analysis showed that one of the bitter receptors, TAS2R38, was expressed in some cells in the small intestine, and immunohistochemical analysis revealed the presence of T2R38-positive cells in the villi of the intestines. (4) Conclusions: These results suggest that bitter receptors are expressed in the gut of rhesus macaques, supporting the use of macaques as a model for studies of human taste, including gut analyses.

Sucralose, an activator of the glucose-sensing receptor, increases ATP by calcium-dependent and -independent mechanisms.

Sucralose is an artificial sweetener and activates the glucose-sensing receptor expressed in pancreatic ß-cells. Although sucralose does not enter ß-cells nor acts as a substrate for glucokinase, it induces a marked elevation of intracellular ATP ([ATP]c). The present study was conducted to identify the signaling pathway responsible for the elevation of [ATP]c induced by sucralose. Previous studies have shown that sucralose elevates cyclic AMP (cAMP), activates phospholipase C (PLC) and stimulates Ca(2+) entry by a Na(+)-dependent mechanism in MIN6 cells. The addition of forskolin induced a marked elevation of cAMP, whereas it did not affect [ATP]c. Carbachol, an activator of PLC, did not increase [ATP]c. In addition, activation of protein kinase C by dioctanoylglycerol did not affect [ATP]c. In contrast, nifedipine, an inhibitor of the voltage-dependent Ca(2+) channel, significantly reduced [ATP]c response to sucralose. Removal of extracellular Na(+) nearly completely blocked sucralose-induced elevation of [ATP]c. Stimulation of Na(+) entry by adding a Na(+) ionophore monensin elevated [ATP]c. The monensin-induced elevation of [ATP]c was only partially inhibited by nifedipine and loading of BAPTA, both of which completely abolished elevation of [Ca(2+)]c. These results suggest that Na(+) entry is critical for the sucralose-induced elevation of [ATP]c. Both calcium-dependent and -independent mechanisms are involved in the action of sucralose.

Hydrogen/deuterium exchange of cross-linkable α-amino acid derivatives in deuterated triflic acid.

In this paper we report here a hydrogen/deuterium exchange (H/D exchange) of cross-linkable α-amino acid derivatives with deuterated trifluoromethanesulfonic acid (TfOD). H/D exchange with TfOD was easily applied to o-catechol containing phenylalanine (DOPA) within an hour. A partial H/D exchange was observed for trifluoromethyldiazirinyl (TFMD) phenylalanine derivatives. N-Acetyl-protected natural aromatic α-amino acids (Tyr and Trp) were more effective in H/D exchange than unprotected ones. The N-acetylated TFMD phenylalanine derivative afforded slightly higher H/D exchange than unprotected derivatives. An effective post-deuteration method for cross-linkable α-amino acid derivatives will be useful for the analysis of biological functions of bioactive peptides and proteins by mass spectrometry.

Continuous flow atmospheric pressure laser desorption/ionization using a 6-7-µm-band mid-infrared tunable laser for biomolecular mass spectrometry.

A continuous flow atmospheric pressure laser desorption/ionization technique using a porous stainless steel probe and a 6-7-µm-band mid-infrared tunable laser was developed. This ion source is capable of direct ionization from a continuous flow with a high temporal stability. The 6-7-µm wavelength region corresponds to the characteristic absorption bands of various molecular vibration modes, including O-H, C=O, CH3 and C-N bonds. Consequently, many organic compounds and solvents, including water, have characteristic absorption peaks in this region. This ion source requires no additional matrix, and utilizes water or acetonitrile as the solvent matrix at several absorption peak wavelengths (6.05 and 7.27 µm, respectively). The distribution of multiply-charged peptide ions is extremely sensitive to the temperature of the heated capillary, which is the inlet of the mass spectrometer. This ionization technique has potential for the interface of liquid chromatography/mass spectrometry (LC/MS).

Elucidating the sequence of intact bioactive peptides by using electron capture dissociation and hot electron capture dissociation in a linear radio-frequency quadrupole ion trap.

RATIONALE: Electron capture dissociation (ECD) is useful tool for sequencing of peptides and proteins with post-translational modifications. To increase the sequence coverage for peptides and proteins, it is important to develop ECD device with high fragmentation efficiency. METHODS: Sequence analysis of intact undigested bioactive peptides (3000-5000 Da) was performed by use of electron capture dissociation (rf-ECD) and collision-induced dissociation (CID) in a linear radio-frequency quadrupole ion trap that was coupled to a time-of-flight mass spectrometer. We applied rf-ECD, hot rf-ECD (rf-ECD with high electron energy), and CID for intact bioactive peptide ions of various charge states and evaluated the sequence coverage of their fragment spectra. RESULTS: Hot rf-ECD produced a higher number of c- and z-type fragment ions of modified peptide ions as electron energy increased in lower charged peptide ions, and sequence coverage greater than 80% was obtained compared with the CID case (40-80%). CONCLUSIONS: The result indicates that intact bioactive modified peptides (Ghrelin, ANP) were correctly identified by use of hot rf-ECD.

Comprehensive synthesis of photoreactive (3-trifluoromethyl)diazirinyl indole derivatives from 5- and 6- trifluoroacetylindoles for photoaffinity labeling.

5- and 6-trifluoromethyldiazirinyl indoles were synthesized from corresponding bromoindole derivatives for the first time. They acted as mother skeletons for the comprehensive synthesis of various bioactive indole metabolites. These can be used in biological functional analysis as diazirine-based photoaffinity labels.

A Data Set of Ion Mobility Collision Cross Sections and Liquid Chromatography Retention Times from 71 Pyridylaminated N-Linked Oligosaccharides.

Determination of the glycan structure is an essential step in understanding structure-function relationships of glycans and glycoconjugates including biopharmaceuticals. Mass spectrometry, because of its high sensitivity and mass resolution, is an excellent means of analyzing glycan structures. We previously proposed a method for rapid and precise identification of N-glycan structures by ultraperformance liquid chromatography-connected ion mobility mass spectrometry (UPLC/IM-MS). To substantiate this methodology, we here examine 71 pyridylaminated (PA-) N-linked oligosaccharides including isomeric pairs. A data set on collision drift times, retention times, and molecular mass was collected for these PA-oligosaccharides. For standardization of the observables, LC retention times were normalized into glucose units (GU) using pyridylaminated α-1,6-linked glucose oligomers as reference, and drift times in IM-MS were converted into collision cross sections (CCS). To evaluate the CCS value of each PA-oligosaccharide, we introduced a CCS index which is defined as a CCS ratio of a target PA-glycan to the putative standard PA-glucose oligomer of the same m/z. We propose a strategy for practical structural analysis of N-linked glycans based on the database of m/z, CCS index, and normalized retention time (GU).

Characterization of the beta-D-glucopyranoside binding site of the human bitter taste receptor hTAS2R16.

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 beta-D-glucopyranoside and will also facilitate rational design of bitter blockers.

Bulky high-mannose-type N-glycan blocks the taste-modifying activity of miraculin.

BACKGROUND: Miraculin (MCL) is a taste-modifying protein that converts sourness into sweetness. The molecular mechanism underlying the taste-modifying action of MCL is unknown. METHODS: Here, a yeast expression system for MCL was constructed to accelerate analysis of its structure-function relationships. The Saccharomyces cerevisiae expression system has advantages as a high-throughput analysis system, but compared to other hosts it is characterized by a relatively low level of recombinant protein expression. To alleviate this weakness, in this study we optimized the codon usage and signal-sequence as the first step. Recombinant MCL (rMCL) was expressed and purified, and the sensory taste was analyzed. RESULTS: As a result, a 2 mg/l yield of rMCL was successfully obtained. Although sensory taste evaluation showed that rMCL was flat in taste under all the pH conditions employed, taste-modifying activity similar to that of native MCL was recovered after deglycosylation. Mutagenetic analysis revealed that the N-glycan attached to Asn42 was bulky in rMCL. CONCLUSIONS: The high-mannose-type N-glycan attached in yeast blocks the taste-modifying activity of rMCL. GENERAL SIGNIFICANCE: The bulky N-glycan attached to Asn42 may cause steric hindrance in the interaction between active residues and the sweet taste receptor hT1R2/hT1R3.

Expression of TAS2R14 in the intestinal endocrine cells of non-human primates.

BACKGROUND: Recent studies have demonstrated that genes related to bitter taste receptors (TAS2Rs) on various chromosomes are expressed in extra-oral organs of various animals. The bitter taste receptor TAS2R14 is conserved among primate species and shows broad ligand sensitivity. Mice have a number of orthologues to primate TAS2R14 located in tandem on chromosome 16; however, their expression patterns are not unique. OBJECTIVE: We characterized the expression of TAS2R14 in various cell types in the intestines of the rhesus macaque and evaluated its role in hormone production in the gut. METHODS: TAS2R14 expression was examined in the intestines of rhesus macaques, a common non-human primate model, by RT-qPCR and immunohistochemical staining. RESULTS: Mean expression levels of TAS2R14 in the duodenum, ileum, and colon were similar to each other and were lower than those in circumvallate papillae. An immunohistochemical analysis revealed TAS2R14 immunoreactivity in enteroendocrine cells positive for cholecystokinin, serotonin, and the G protein GNAT3. CONCLUSION: These results suggest that primate TAS2R14 is broadly expressed in the intestine, mainly in enteroendocrine cells, and promotes gut hormone secretion in response to bitter stimuli.

Photoactive ligands probing the sweet taste receptor. Design and synthesis of highly potent diazirinyl D-phenylalanine derivatives.

Some D-amino acids such as d-tryptophan and D-phenylalanine are well known as naturally-occurring sweeteners. Photoreactive D-phenylalanine derivatives containing trifluoromethyldiazirinyl moiety at 3- or 4-position of phenylalanine, were designed as sweeteners for functional analysis with photoaffinity labeling. The trifluoromethyldiazirinyl D-phenylalanine derivatives were prepared effectively with chemo-enzymatic methods using L-amino acid oxidase and were found to have potent activity toward the human sweet taste receptor.

Correction to: Cell-free synthesis of functionally active HSPB5.

In the original publication, the given name of the last author was incorrectly displayed as the name must read: Katsuyoshi Masuda.

Hydrophobic interactions at subsite S1' of human dipeptidyl peptidase IV contribute significantly to the inhibitory effect of tripeptides.

Functional inhibitory peptides of human dipeptidyl peptidase 4 (hDPP4) have been highly anticipated as the active ingredient of functional food for type II diabetes; however, the molecular mechanism of hDPP4 inhibition remains unclear. In this study, we focused on dipeptides and tripeptides, which display structure-function correlations that are relatively easy to analyze, and examined their interactions with hDPP4 on an atomic level using a combination of docking studies and an hDPP4 inhibition assay. First, we performed comprehensive binding mode analysis of the dipeptide library and demonstrated that the formation of a tight interaction with the S1 subsite composing part of the substrate pocket is essential for dipeptides to compete with the substrate and strongly inhibit hDPP4. Next, we synthesized tripeptides by adding various amino acids to the C-terminus of Ile-Pro and Val-Pro, which have especially high inhibitory activity among compounds in the dipeptide library, and measured the hDPP4 inhibitory activity of the tripeptides. When hydrophobic amino acids (Ile, Met, Val, Trp) were added, the inhibitory activity increased several-fold. This phenomenon could be explained as follows: the C-terminal amino acid of the tripeptide formed hydrophobic interactions with Tyr547 and Trp629, which compose the S1' subsite located relatively outside the substrate pocket, thereby stabilizing the hDPP4-peptide binding. The structural information on the interaction between hDPP4 and peptide inhibitors attained in this study is anticipated to be useful in the development of a more potent hDPP4 competitive inhibitor.

Analogue chromophore study of the influence of electronic perturbation on color regulation of photoactive yellow protein.

We report a unique lambdamax shift of the absorption maximum of a photoactive yellow protein (PYP) analogue reconstituted with a fluorinated chromophore (F-PYP). The difference in lambdamax between the free chromophore and the protein was significantly larger than that with the native chromophore. We concluded that the unusual lambdamax shift is caused by the electronegative character of the fluorine atom and not by steric hindrance. This result suggests that formation of a hydrogen bond between the fluorine atom and one or more amino acid residues could neutralize its electron-withdrawing character. The properties of analogues of PYP with brominated and methylated chromophore could be explained as an effect of steric hindrance.

Amino acid residues of bitter taste receptor TAS2R16 that determine sensitivity in primates to ß-glycosides.

In mammals, bitter taste is mediated by TAS2Rs, which belong to the family of seven transmembrane G protein-coupled receptors. Since 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 analyzed the amino acids responsible for the difference in sensitivities of TAS2R16s of various primates using a cultured cell expression system. We found that the sensitivity of TAS2R16 varied due to several amino acid residues. Mutation of amino acid residues at E86T, L247M, and V260F in human and langur TAS2R16 for mimicking the macaque TAS2R16 decreased the sensitivity of the receptor in an additive manner, which suggests its contribution to the potency of salicin, possibly via direct interaction. However, mutation of amino acid residues 125 and 133 in human TAS2R16, which are situated in helix 4, to the macaque sequence increased the sensitivity of the receptor. 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 species-specific food.

Analysis of neutral oligosaccharides for structural characterization by matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometry.

We have acquired multi-stage mass spectra (MSn) of four branched N-glycans derived from human serum IgG by matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometry (MALDI-QIT-TOF-MS) in order to demonstrate high sensitivity structural analysis. [M+H]+ and [M+Na]+ ions were detected in the positive mode. The detection limit of [M+Na]+ in MS/MS and MS3 measurements for structural analysis was found to be 100 fmol, better than that for [M+H]+. The [M+H]+ ions subsequently fragmented to produce predominantly a Y series of fragments, whereas [M+Na]+ ions fragmented to give a complex mixture of B and Y ions together with some cross-ring fragments. Three features of MALDI-QIT-CID fragmentation of [M+Na]+ were cleared by the analysis of MS/MS, MS3 and MS4 spectra: (1) the fragment ions resulting from the breaking of a bond are more easily generated than that from multi-bond dissociation; (2) the trimannosyl-chitobiose core is either hardly dissociated, easily ionized or it is easy to break a bond between N-acetylglucosamine and mannose; (3) the fragmentation by loss of only galactose from the non-reducing terminus is not observed. We could determine the existence ratios of candidates for each fragment ion in the MS/MS spectrum of [M+Na]+ by considering these features. These results indicate that MSn analysis of [M+Na]+ ions is more useful for the analysis of complicated oligosaccharide structures than MS/MS analysis of [M+H]+, owing to the higher sensitivity and enhanced structural information. Furthermore, two kinds of glycans, with differing branch structures, could be distinguished by comparing the relative fragment ion abundances in the MS3 spectrum of [M+Na]+. These analyses demonstrate that the MSn technology incorporated in MALDI-QIT-TOF-MS can facilitate the elucidation of structure of complex branched oligosaccharides.

Differences in properties between human alphaA- and alphaB-crystallin proteins expressed in Escherichia coli cells in response to cold and extreme pH.

It has been reported that alphaA-crystallin has greater protective effects against apoptosis in lens epithelial cells than alphaB-crystallin [Andley, Song, Wawrousek, Fleming and Bassnett (2000) J. Biol. Chem. 275, 36823-36831]. Because the alphaA-crystallin proteins are specifically expressed in the vertebrate lens, we examine the non-specific properties of both alphaA- and alphaB-crystallins in an Escherichia coli system. E. coli cells were transformed with the inducible protein expression vector pET-11a, harbouring the gene for either human alphaA- or alphaB-crystallin, and two other control plasmids, pET-1la vector alone or pGEX-2T vector encoding GST (glutathione S-transferase). These cells were exposed to various stress conditions, such as cold-shock at 4 degrees C or extremely low or high pH environments (pH 4.7 or pH 8.0) for 6 h, and survival of the host cells and the solubility of the expressed target proteins in the cytosol were examined. Under these stress conditions, the cells expressing alphaB-crystallin protein demonstrated significantly improved survival when compared with the other cells, and the expressed protein in the cytosol was almost soluble, in contrast with the alphaA-crystallin protein. Differences in the amino acid sequence between the proteins in a phenylalanine-rich region next to the N-terminal consensus alpha-crystallin domain was considered to be responsible for chaperone activity and cell survival.

Atmospheric pressure laser desorption/ionization using a 6-7 µm-band mid-infrared tunable laser and liquid water matrix.

Due to the characteristic absorption peaks in the IR region, various molecules can be used as a matrix for infrared matrix-assisted laser desorption/ionization (IR-MALDI). Especially in the 6-7 µm-band IR region, solvents used as the mobile phase for liquid chromatography have absorption peaks that correspond to their functional groups, such as O-H, C=O, and CH3. Additionally, atmospheric pressure (AP) IR-MALDI, which is applicable to liquid-state samples, is a promising technique to directly analyze untreated samples. Herein we perform AP-IR-MALDI mass spectrometry of a peptide, angiotensin II, using a mid-IR tunable laser with a tunable wavelength range of 5.50-10.00 µm and several different matrices. The wavelength dependences of the ion signal intensity of [M + H](+) of the peptide are measured using a conventional solid matrix, α-cyano-4-hydroxycinnamic acid (CHCA) and a liquid matrix composed of CHCA and 3-aminoquinoline. Other than the O-H stretching and bending vibration modes, the characteristic absorption peaks are useful for AP-IR-MALDI. Peptide ions are also observed from an aqueous solution of the peptide without an additional matrix, and the highest peak intensity of [M + H](+) is at 6.00 µm, which is somewhat shorter than the absorption peak wavelength of liquid water corresponding to the O-H bending vibration mode. Moreover, long-lasting and stable ion signals are obtained from the aqueous solution. AP-IR-MALDI using a 6-7 µm-band IR tunable laser and solvents as the matrix may provide a novel on-line interface between liquid chromatography and mass spectrometry.