RESUMO
A study of the structural requirements of cholic acid derivatives as liverâ¯Xâ¯receptor (LXR) ligands was performed. A model of cholenamide derivative 1 complexed with LXR showed that the C24 carbonyl oxygen forms a hydrogen bond with His435 located close to Trp457. The N,N-dimethyl group is located in a hydrophobic pocket. Based on these data, we designed compounds with high affinity for LXRs. Cholenamide derivatives 1-11 were synthesized from 3ß-acetyl-Δ5-cholenic acid 20, and lactams 12-19 were synthesized from alcohol 25. Tertiary amides 3 and 4 showed higher activity in reporter assays, and compounds with hydrophobic residues exhibited the highest activity of all derivatives. The stereochemistry at C23 was found to be an important determinant of EC50 and gene transactivation, as each isomer exhibited different activity.
Assuntos
Amidas/farmacologia , Ácido Cólico/farmacologia , Receptores X do Fígado/metabolismo , Amidas/síntese química , Amidas/química , Animais , Ácido Cólico/síntese química , Ácido Cólico/química , Relação Dose-Resposta a Droga , Humanos , Ligantes , Camundongos , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
Novel carbohydrate mimics were designed which contain two 5a-carba-d-glucose residues, one each at reducing and nonreducing end, and thus these mimics are 5a,5a'-dicarba-d-glucobioses. Dicarbadisaccharides have attractive features such as stability against endogenous degradative enzymes and being resistant to glycation reactions such as the Maillard reaction. For the synthesis of dicarba-ß-d-isomaltose derivatives, the carbaglucosyl triflate locked in 4C1 conformation was synthesized by protecting with butane-2,3-diacetal group or benzylidene group. Then, 5a,5a'-dicarba-ß-d-maltose and 5a,5a'-dicarba-α,ß-d-trehalose were synthesized by the SN2-type inversion reaction using 4,6-O-benzylidene carbaglucosyl triflate with 4-OH and 1-OH carba-ß-d-glucose derivatives, respectively, and similarly 5a,5a'-dicarba-α-d-isomaltose with 6-OH carba-α-d-glucose derivative.
Assuntos
Cicloexanóis/síntese química , Dissacarídeos/síntese química , Mesilatos/química , Conformação MolecularRESUMO
TGR5, a member of the G protein-coupled receptor (GPCR) family, is activated by bile acids. Because TGR5 promotes energy expenditure and improves glucose homeostasis, it is recognized as a key target in treating metabolic diseases. We previously showed that nomilin, a citrus limonoid, activates TGR5 and confers anti-obesity and anti-hyperglycemic effects in mice. Information on the TGR5-nomilin interaction regarding molecular structure, however, has not been reported. In the present study, we found that human TGR5 (hTGR5) shows higher nomilin responsiveness than does mouse TGR5 (mTGR5). Using mouse-human chimeric TGR5, we also found that three amino acid residues (Q77ECL1, R80ECL1, and Y893.29) are important in the hTGR5-nomilin interaction. Based on these results, an hTGR5-nomilin binding model was constructed using in silico docking simulation, demonstrating that four hydrophilic hydrogen-bonding interactions occur between nomilin and hTGR5. The binding mode of hTGR5-nomilin is vastly different from those of other TGR5 agonists previously reported, suggesting that TGR5 forms various binding patterns depending on the type of agonist. Our study promotes a better understanding of the structure of TGR5, and it may be useful in developing and screening new TGR5 agonists.
Assuntos
Aminoácidos/química , Benzoxepinas/química , Benzoxepinas/metabolismo , Limoninas/química , Limoninas/metabolismo , Modelos Moleculares , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Sequência de Aminoácidos , Animais , Células HEK293 , Humanos , Camundongos , Simulação de Acoplamento Molecular , Mutação/genética , Especificidade por Substrato , Ácido Taurolitocólico/metabolismoRESUMO
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.
RESUMO
Among proteins utilized as sweeteners, neoculin and miraculin are taste-modifying proteins that exhibit pH-dependent sweetness. Several experiments on neoculin have shown that His11 of neoculin is responsible for pH dependence. We investigated the molecular mechanism of the pH dependence of neoculin by molecular dynamics (MD) calculations. The MD calculations for the dimeric structures of neoculin and His11 mutants showed no significant structural changes for each monomer at neutral and acidic pH levels. The dimeric structure of neoculin dissociated to form isolated monomers under acidic conditions but was maintained at neutral pH. The dimeric structure of the His11Ala mutant, which is sweet at both neutral and acidic pH, showed dissociation at both pH 3 and 7. The His11 residue is located at the interface of the dimer in close proximity to the Asp91 residue of the other monomer. The MD calculations for His11Phe and His11Tyr mutants demonstrated the stability of the dimeric structures at neutral pH and the dissociation of the dimers to isolated monomers. The dissociation of the dimer caused a flexible backbone at the surface that was different from the dimeric interface at the point where the other monomer interacts to form an oligomeric structure. Further MD calculations on the tetrameric structure of neoculin suggested that the flexible backbone contributed to further dissociation of other monomers under acidic conditions. These results suggest that His11 plays a role in the formation of oligomeric structures at pH 7 and that the isolated monomer of neoculin at acidic pH is responsible for sweetness.
Assuntos
Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Paladar , Aminoácidos/química , Cristalografia por Raios X , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Simulação de Dinâmica Molecular , Proteínas Mutantes/química , Mutação/genética , Multimerização ProteicaRESUMO
Natural sterols often occur as a heterogeneous mixture of homologs, which had disturbed the progress of steroid research. Development and application of GC methodology overcame this difficulty and enabled us to obtain detailed sterol profiles. Together, fine synthesis of stereo-defined isomers and homologs of steroids having oxygenated side chains allowed us to compare them with natural samples as well as to investigate structure-activity relationship. Advance of HPLC technology also facilitated the determination of the stereochemical structure of naturally occurring steroidal compounds, which were obtained only in minute amounts. This review highlights three topics out of our steroid research that have been performed mainly at Tokyo Institute of Technology around 1970-1990. These are sterol metabolism in insects focusing on the mechanism of the conversion of plant sterols to cholesterol and ecdysone biosynthesis, the synthesis and biochemical research of active forms of vitamin D3 derivatives, and the synthesis and microanalysis of plant hormone brassinosteroids.
Assuntos
Insetos/metabolismo , Plantas/metabolismo , Pesquisa , Esteróis/química , Esteróis/metabolismo , Animais , Brassinosteroides/química , Brassinosteroides/metabolismo , Colecalciferol/química , Colecalciferol/metabolismo , Humanos , Esteróis/biossínteseRESUMO
BACKGROUND AND PURPOSE The free fatty acid FFA1 receptor and GPR120 are GPCRs whose endogenous ligands are medium- and long-chain FFAs, and they are important in regulating insulin and GLP-1 secretion respectively. Given that the ligands of FFA1 receptor and GPR120 have similar properties, selective pharmacological tools are required to study their functions further. EXPERIMENTAL APPROACH We used a docking simulation approach using homology models for each receptor. Biological activity was assessed by phosphorylation of ERK and elevation of intracellular calcium ([Ca(2+) ]i ) in cells transfected with FFA1 receptor or GPR120. Insulin secretion from murine pancreatic beta cells (MIN6) was also measured. KEY RESULTS Calculated hydrogen bonding energies between a series of synthetic carboxylic acid compounds and the homology models of the FFA1 receptor and GPR120, using docking simulations, correlated well with the effects of the compounds on ERK phosphorylation in transfected cells (R(2) = 0.65 for FFA1 receptor and 0.76 for GPR120). NCG75, the compound with the highest predicted selectivity for FFA1 receptors from this structure-activity relationship analysis, activated ERK and increased [Ca(2+) ]i as potently as the known FFA1 receptor-selective agonist, Compound 1. Site-directed mutagenesis analysis based on the docking simulation showed that different amino acid residues were important for the recognition and activation by FFA1 receptor agonists. Moreover, NCG75 strongly induced ERK and [Ca(2+) ]i responses, and promoted insulin secretion from MIN6 cells, which express endogenous FFA1 receptors. CONCLUSION AND IMPLICATIONS A docking simulation approach using FFA1 receptor and GPR120 homology models could be useful in predicting FFA1 receptor-selective agonists.
Assuntos
Ácidos Graxos não Esterificados/metabolismo , Ácidos Láuricos/química , Simulação de Acoplamento Molecular , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/química , Animais , Cálcio/metabolismo , Linhagem Celular , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Ligação de Hidrogênio , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Ácidos Láuricos/farmacologia , Ligantes , Camundongos , Mutagênese Sítio-Dirigida , Fosforilação , Receptores Acoplados a Proteínas G/genética , Relação Estrutura-AtividadeRESUMO
We previously reported that sarpogrelate, a selective 5-HT2A antagonist, showed a potent inverse agonist activity to constitutively active mutant (C322K) of human 5-HT2A receptor (5-HT2AR). However, it remains to be unknown about the actual mechanism of this mutant for its constitutive activation as well as inverse agonist activity of sarpogrelate. Our model shows that mutation (C322K) of 5-HT2AR causes electronic repulsion between positively charged Arg173(3.50) and Lys322(6.34) residues resulting outward movement of the C-terminus of transmembrane helix (TMH) III. This motion of TMH III leads to a partially active structure of the receptor, which may be a key step in receptor activation. The structural model of the partially active receptor also indicates that the binding of sarpogrelate to the constitutively active receptor causes an inward swing of TMH III to an inactive receptor structure. Therefore, the present study may suggest that the electronic repulsion causing outward movement of the C-terminus of TMH III may be the key step for constitutive activation of mutant C322K of 5-HT2AR and the inward movement of TMH III causes the inverse agonist activity of sarpogrelate.
Assuntos
Mutação , Receptor 5-HT2A de Serotonina/metabolismo , Antagonistas do Receptor 5-HT2 de Serotonina/farmacologia , Succinatos/farmacologia , Células HEK293 , Humanos , Modelos Moleculares , Estrutura Secundária de Proteína , Receptor 5-HT2A de Serotonina/química , Receptor 5-HT2A de Serotonina/genéticaRESUMO
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.
Assuntos
Receptores Acoplados a Proteínas G/agonistas , Edulcorantes/química , Motivos de Aminoácidos , Substituição de Aminoácidos , Aspartame/química , Sítios de Ligação , Simulação por Computador , Ciclamatos/química , Ciclamatos/farmacologia , Células HEK293 , Humanos , Modelos Moleculares , Peso Molecular , Mutagênese Sítio-Dirigida , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Sacarina/química , Sacarina/farmacologia , Sacarose/análogos & derivados , Sacarose/química , Sacarose/farmacologia , Edulcorantes/farmacologia , Tiazinas/química , Tiazinas/farmacologia , Triptofano/química , Triptofano/farmacologiaRESUMO
Despite many recent studies of G-protein-coupled receptor (GPCR) structures, it is not yet well understood how these receptors activate G proteins. The GPCR assay using baker's yeast, Saccharomyces cerevisiae, is an effective experimental model for the characterization of GPCR-Gα interactions. Here, using the yeast endogenous Gα protein (Gpa1p) as template, we constructed various chimeric Gα proteins with a region that is considered to be necessary for interaction with mammalian receptors. The signaling assay using the yeast pheromone receptor revealed that the chimeric Gα protein harboring 37 gustducin-specific amino acid residues at its C-terminus (GPA1/gust37) maintained functionality in yeast. In contrast, GPA1/gust44, a variant routinely used in mammalian experimental systems, was not functional.
Assuntos
Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Engenharia Genética/métodos , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Transducina/genética , Sequência de Aminoácidos , Subunidades alfa de Proteínas de Ligação ao GTP/química , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Pseudomonas aeruginosa, an opportunistic pathogen, is known to mainly use N-acylhomoserine lactones (AHLs) as autoinducers. Recent progress in this field demonstrated that not only AHLs, but also their degradation products, tetramic acids, may have some biological activities. The present study examined the roles of Pseudomonas autoinducers and tetramic acids in bacterial survival and behavior in ecological niches. P. aeruginosa autoinducers and the tetramic acid derivatives were chemically synthesized, and the structure-activity correlation was investigated. Some tetramic acids derived from AHLs caused a significant reduction in the viability of P. aeruginosa in a concentration dependent manner (30-300 µM). The smaller the inoculum of bacteria, the stronger the bactericidal activity that was observed. The data from tetramic acid derivatives indicated the keto-enol structure of tetramic acid to be critical for the antibacterial activity. Exogenous tetramic acid did not induce significant changes in the formation of biofilm or production of exoproducts such as pyocyanin and elastase. Tetramic acid and disinfectants acted synergistically to kill P. aeruginosa. These results suggest the AHL-degradation product tetramic acid to be useful for bacterial control.
Assuntos
Antibacterianos/farmacologia , Homosserina/análogos & derivados , Pseudomonas aeruginosa/metabolismo , Acil-Butirolactonas/metabolismo , Desinfetantes/farmacologia , Escherichia coli/crescimento & desenvolvimento , Homosserina/metabolismo , Homosserina/fisiologia , Klebsiella pneumoniae/crescimento & desenvolvimento , Lactonas/metabolismo , Viabilidade Microbiana/efeitos dos fármacos , Estrutura Molecular , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pirrolidinonas/farmacologia , Percepção de Quorum/fisiologia , Relação Estrutura-AtividadeRESUMO
We investigated a formation channel of triatomic molecular hydrogen ions from ethane dication induced by irradiation of intense laser fields (800 nm, 100 fs, â¼1 × 10(14) W∕cm(2)) by using time of flight mass spectrometry. Hydrogen ion and molecular hydrogen ion (H,D)(n)(+) (n = 1-3) ejected from ethane dications, produced by double ionization of three types of samples, CH(3)CH(3), CD(3)CD(3), and CH(3)CD(3), were measured. All fragments were found to comprise components with a kinetic energy of â¼3.5 eV originating from a two-body Coulomb explosion of ethane dications. Based on the signal intensities and the anisotropy of the ejection direction with respect to the laser polarization direction, the branching ratios, H(+):D(+) = 66:34, H(2)(+):HD(+):D(2)(+) = 63:6:31, and H(3)(+):H(2)D(+):HD(2)(+):D(3)(+) = 26:31:34:9 for the decomposition of C(2)H(3)D(3)(2+), were determined. The ratio of hydrogen molecules, H(2):HD:D(2) = 31:48:21, was also estimated from the signal intensities of the counter ion C(2)(H,D)(4)(2+). The similarity in the extent of H∕D mixture in (H,D)(3)(+) with that of (H,D)(2) suggests that these two dissociation channels have a common precursor with the C(2)H(4)(2+)...H(2) complex structure, as proposed theoretically in the case of H(3)(+) ejection from allene dication [A. M. Mebel and A. D. Bandrauk, J. Chem. Phys. 129, 224311 (2008)]. In contrast, the (H,D)(2)(+) ejection path with a lower extent of H∕D mixture and a large anisotropy is expected to proceed essentially via a different path with a much rapid decomposition rate. For the Coulomb explosion path of C-C bond breaking, the yield ratios of two channels, CH(3)CD(3)(2+)â CH(3)(+) + CD(3)(+) and CH(2)D(+) + CHD(2)(+), were 81:19 and 92:8 for the perpendicular and parallel directions, respectively. This indicates that the process occurs at a rapid rate, which is comparable to hydrogen migration through the C-C bond, resulting in smaller anisotropy for the latter channel that needs H∕D exchange.
Assuntos
Etano/química , Hidrogênio/química , Lasers , Cátions/química , Fatores de TempoRESUMO
Sweetness and bitterness are key determinants of food acceptance and rejection, respectively. Sugars, such as sucrose and fructose, are generally recognized as sweet. However, not all sugars are sweet, and even anomers may have quite different tastes. For example, gentiobiose is bitter, whereas its anomer, isomaltose, is sweet. Despite this unique sensory character, the molecular basis of the bitterness of gentiobiose remains to be clarified. In this study, we used calcium imaging analysis of human embryonic kidney 293T cells that heterologously expressed human taste receptors to demonstrate that gentiobiose activated hTAS2R16, a bitter taste receptor, but not hT1R2/hT1R3, a sweet taste receptor. In contrast, isomaltose activated hT1R2/hT1R3. As a result, these anomers elicit different taste sensations. Mutational analysis of hTAS2R16 also indicated that gentiobiose and ß-D-glucopyranosides, such as salicin share a common binding site of hTAS2R16.
Assuntos
Dissacarídeos/química , Receptores Acoplados a Proteínas G/metabolismo , Paladar/fisiologia , Álcoois Benzílicos/química , Sítios de Ligação , Linhagem Celular , Dissacarídeos/metabolismo , Glucosídeos/química , Humanos , Conformação Molecular , Mutação , Receptores Acoplados a Proteínas G/genéticaRESUMO
We identified two glycosyltransferases that contribute to the structural diversification of flavonol glycosides in grapevine (Vitis vinifera): glycosyltransferase 5 (Vv GT5) and Vv GT6. Biochemical analyses showed that Vv GT5 is a UDP-glucuronic acid:flavonol-3-O-glucuronosyltransferase (GAT), and Vv GT6 is a bifunctional UDP-glucose/UDP-galactose:flavonol-3-O-glucosyltransferase/galactosyltransferase. The Vv GT5 and Vv GT6 genes have very high sequence similarity (91%) and are located in tandem on chromosome 11, suggesting that one of these genes arose from the other by gene duplication. Both of these enzymes were expressed in accordance with flavonol synthase gene expression and flavonoid distribution patterns in this plant, corroborating their significance in flavonol glycoside biosynthesis. The determinant of the specificity of Vv GT5 for UDP-glucuronic acid was found to be Arg-140, which corresponded to none of the determinants previously identified for other plant GATs in primary structures, providing another example of convergent evolution of plant GAT. We also analyzed the determinants of the sugar donor specificity of Vv GT6. Gln-373 and Pro-19 were found to play important roles in the bifunctional specificity of the enzyme. The results presented here suggest that the sugar donor specificities of these Vv GTs could be determined by a limited number of amino acid substitutions in the primary structures of protein duplicates, illustrating the plasticity of plant glycosyltransferases in acquiring new sugar donor specificities.
Assuntos
Flavonoides/química , Glicosídeos/química , Glicosiltransferases/metabolismo , Vitis/enzimologia , Sequência de Aminoácidos , Glicosiltransferases/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Filogenia , RNA de Plantas/genética , Alinhamento de Sequência , Especificidade por Substrato , Vitis/genéticaRESUMO
GPR120 is a G protein-coupled receptor expressed preferentially in the intestinal tract and adipose tissue, that has been implicated in mediating free fatty acid-stimulated glucagon-like peptide-1 (GLP-1) secretion. To develop GPR120-specific agonists, a series of compounds (denoted as NCG compounds) derived from a peroxisome proliferator-activated receptor γ agonist were synthesized, and their structure-activity relationships as GPR120 agonists were explored. To examine the agonistic activities of these newly synthesized NCG compounds, and of compounds already shown to have GPR120 agonistic activity (grifolic acid and MEDICA16), we conducted docking simulation in a GPR120 homology model that was developed on the basis of a photoactivated model derived from the crystal structure of bovine rhodopsin. We calculated the hydrogen bonding energies between the compounds and the GPR120 model. These energies correlated well with the GPR120 agonistic activity of the compounds (R(2) = 0.73). NCG21, the NCG compound with the lowest calculated hydrogen bonding energy, showed the most potent extracellular signal-regulated kinase (ERK) activation in a cloned GPR120 system. Furthermore, NCG21 potently activated ERK, intracellular calcium responses and GLP-1 secretion in murine enteroendocrine STC-1 cells that express GPR120 endogenously. Moreover, administration of NCG21 into the mouse colon caused an increase in plasma GLP-1 levels. Taken together, our present study showed that a docking simulation using a GPR120 homology model might be useful to predict the agonistic activity of compounds.
Assuntos
Aminopiridinas/química , Modelos Moleculares , Fenilbutiratos/química , Receptores Acoplados a Proteínas G/agonistas , Sequência de Aminoácidos , Aminopiridinas/farmacologia , Animais , Sítios de Ligação , Cálcio/metabolismo , Bovinos , Linhagem Celular , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Ligação de Hidrogênio , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Fenilbutiratos/farmacologia , Fosforilação , Ligação Proteica , Receptores Acoplados a Proteínas G/química , Homologia de Sequência de Aminoácidos , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Glucosídeos/metabolismo , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Álcoois Benzílicos/metabolismo , Sítios de Ligação , Linhagem Celular , Humanos , Ligantes , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação Puntual , Ligação Proteica , Conformação Proteica , Receptores Acoplados a Proteínas G/genética , Especificidade por SubstratoRESUMO
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.
Assuntos
Glicoproteínas , Polissacarídeos , Receptores Acoplados a Proteínas G/agonistas , Proteínas Recombinantes , Paladar/efeitos dos fármacos , Glicoproteínas/biossíntese , Glicoproteínas/genética , Glicoproteínas/farmacologia , Humanos , Polissacarídeos/biossíntese , Polissacarídeos/genética , Polissacarídeos/farmacologia , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Saccharomyces cerevisiae/genética , Paladar/fisiologiaRESUMO
The present study was designed to examine the binding affinity and functional potency of selective angiotensin II type 1 (AT(1))-receptor antagonists towards specific mutants of AT(1) receptor using site-directed mutagenesis. We also compared our results with the wild-type AT(1) receptor and investigated the possible reasons behind that. Both wild-type and mutant receptors were expressed in COS-7 cells and the binding affinities of the antagonists were determined by radioligand binding assay. Inhibition of agonist-stimulated inositol phosphate accumulation by the antagonists was also done. Substitution of asparagine(235) of intracellular loop 3 of the AT(1) receptor by arginine increased the binding affinity of the antagonists 5 - 34-fold, whereas the increase in the binding affinity of the antagonists in the phenylalanine(239) mutant by arginine and tryptophan (F239R and F239W) were 3 - 19-fold and 2 - 15-fold higher, respectively, compared to the wild-type AT(1) receptor. The results suggested that substitution by a positively charged or sterically hindered amino acid in the AT(1) receptor allows it to interact with the acidic tetrazole moiety and carboxylate groups of the antagonists more strongly compared to the wild-type receptor. These findings may play an important role to change the binding affinity of the antagonists to an effective level for the pharmacological function of the drugs.
Assuntos
Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Mutagênese Sítio-Dirigida/métodos , Receptor Tipo 1 de Angiotensina/efeitos dos fármacos , Receptor Tipo 1 de Angiotensina/genética , Substituição de Aminoácidos , Animais , Sítios de Ligação , Células COS , Chlorocebus aethiops , Interações Medicamentosas , Fosfatos de Inositol/metabolismo , Ensaio Radioligante , Receptor Tipo 1 de Angiotensina/agonistasRESUMO
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.
Assuntos
Fenilalanina/síntese química , Fenilalanina/metabolismo , Marcadores de Fotoafinidade/síntese química , Marcadores de Fotoafinidade/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Papilas Gustativas/metabolismo , Linhagem Celular , Cristalografia por Raios X , Humanos , Estrutura Terciária de Proteína , Receptores Acoplados a Proteínas G/química , Edulcorantes/metabolismo , Papilas Gustativas/químicaRESUMO
We have examined the potential bactericidal activities of several tetramic acids derived from Pseudomonas autoinducers against Clostridium difficile, a cause of antibiotic-associated pseudomembranous colitis. Clinical isolates of C. difficile (n=4) were incubated in broth with a chemically synthesized Pseudomonas autoinducer and its tetramic acid derivatives. The structure-activity relationship and the mechanisms of action were examined by a time-killing assay and by determination of the morphological/staining characteristics. The use of some tetramic acids derived from N-3-oxododecanoyl L-homoserine lactone resulted in more than 3-log reductions in the viability of C. difficile within 30 min at 30 microM. The outer membrane was suggested to be one of the targets for the bactericidal activity of tetramic acid, because disturbance of the bacterial outer surface was demonstrated by alteration of the Gram-staining characteristic and electron microscopy. The data for the tetramic acid derivatives demonstrate that the keto-enol structure and the length of the acyl side chain of tetramic acid may be essential for the antibacterial activity of this molecule. These results suggest the potential for tetramic acid derivatives to be novel agents with activity against C. difficile.