RESUMO
C-2 fluorinated and methylated stereoisomers of the fragrance citronellol 1 and its oxalate esters were prepared from (R)-pulegone 11 and explored as agonists of the human olfactory receptor OR1A1 and assayed also against site-specific mutants. There were clear isomer preferences and C-2 difluorination as in 18 led to the most active compound suggesting an important hydrogen bond donor role for citronellol 1. C-2 methylation and the corresponding oxalate ester analogues were less active.
Assuntos
Monoterpenos Acíclicos , Receptores Odorantes , Monoterpenos Acíclicos/química , Ésteres/química , Humanos , Ligação de Hidrogênio , Oxalatos , Receptores Odorantes/agonistasRESUMO
Aroma is an important attribute influencing the perceived quality of Chinese liquors, with each liquor characterized by a unique collection of volatile chemicals. Here, a biomimetic olfactory recognition system combining an optimal panel of 10 mouse odorant receptors with back propagation neural network model was designed to discriminate the aromas of Chinese liquors. Our system shows an excellent predictive capacity with an average accuracy of 96.5% to discriminate liquors of different aroma types, as well as those of different brands and ageing years within the same aroma type. A total of 124 interactions between liquor aroma compounds and odorant receptors were further elucidated to understand odorant coding at the molecular level, including 14 newly deorphaned odorant receptors. Our work represents a proof of concept for combining receptors and machine learning in the discrimination of complex odorant stimuli.
Assuntos
Bebidas Alcoólicas , Biomimética , Odorantes , Receptores Odorantes , Bebidas Alcoólicas/análise , Animais , China , Aprendizado de Máquina , Camundongos , Redes Neurais de Computação , Odorantes/análiseRESUMO
Metal-coordinating compounds are generally known to have strong smells, a phenomenon that can be attributed to the fact that odorant receptors for intense-smelling compounds, such as those containing sulfur, may be metalloproteins. We previously identified a mouse odorant receptor (OR), Olfr1509, that requires copper ions for sensitive detection of a series of metal-coordinating odorants, including (methylthio)methanethiol (MTMT), a strong-smelling component of male mouse urine that attracts female mice. By combining mutagenesis and quantum mechanics/molecular mechanics (QM/MM) modeling, we identified candidate binding sites in Olfr1509 that may bind to the copper-MTMT complex. However, whether there are other receptors utilizing metal ions for ligand-binding and other sites important for receptor activation is still unknown. In this study, we describe a second mouse OR for MTMT with a copper effect, namely Olfr1019. In an attempt to investigate the functional changes of metal-coordinating ORs in multiple species and to decipher additional sites involved in the metal effect, we cloned various mammalian orthologs of the 2 mouse MTMT receptors, and a third mouse MTMT receptor, Olfr15, that does not have a copper effect. We found that the function of all 3 MTMT receptors varies greatly among species and that the response to MTMT always co-occurred with the copper effect. Furthermore, using ancestral reconstruction and QM/MM modeling combined with receptor functional assay, we found that the amino acid residue R260 in Olfr1509 and the respective R261 site in Olfr1019 may be important for receptor activation.
Assuntos
Cobre/metabolismo , Receptores Odorantes/metabolismo , Compostos de Sulfidrila/química , Sulfetos/química , Animais , Gatos , Bovinos , Cobre/química , Cricetulus , Cães , Cobaias , Cavalos , Teoria Quântica , Coelhos , Ratos , Ratos Sprague-Dawley , SuínosRESUMO
Understanding olfaction at the molecular level is challenging due to the lack of crystallographic models of odorant receptors (ORs). To better understand the molecular mechanism of OR activation, we focused on chiral (R)-muscone and other musk-smelling odorants due to their great importance and widespread use in perfumery and traditional medicine, as well as environmental concerns associated with bioaccumulation of musks with estrogenic/antiestrogenic properties. We experimentally and computationally examined the activation of human receptors OR5AN1 and OR1A1, recently identified as specifically responding to musk compounds. OR5AN1 responds at nanomolar concentrations to musk ketone and robustly to macrocyclic sulfoxides and fluorine-substituted macrocyclic ketones; OR1A1 responds only to nitromusks. Structural models of OR5AN1 and OR1A1 based on quantum mechanics/molecular mechanics (QM/MM) hybrid methods were validated through direct comparisons with activation profiles from site-directed mutagenesis experiments and analysis of binding energies for 35 musk-related odorants. The experimentally found chiral selectivity of OR5AN1 to (R)- over (S)-muscone was also computationally confirmed for muscone and fluorinated (R)-muscone analogs. Structural models show that OR5AN1, highly responsive to nitromusks over macrocyclic musks, stabilizes odorants by hydrogen bonding to Tyr260 of transmembrane α-helix 6 and hydrophobic interactions with surrounding aromatic residues Phe105, Phe194, and Phe207. The binding of OR1A1 to nitromusks is stabilized by hydrogen bonding to Tyr258 along with hydrophobic interactions with surrounding aromatic residues Tyr251 and Phe206. Hydrophobic/nonpolar and hydrogen bonding interactions contribute, respectively, 77% and 13% to the odorant binding affinities, as shown by an atom-based quantitative structure-activity relationship model.
Assuntos
Cicloparafinas/química , Modelos Moleculares , Receptores Odorantes/química , Células HEK293 , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Mutagênese Sítio-Dirigida , Estabilidade Proteica , Estrutura Secundária de Proteína , Receptores Odorantes/genética , Receptores Odorantes/metabolismoRESUMO
The enormous sizes of the mammalian odorant receptor (OR) families present difficulties to find their cognate ligands among numerous volatile chemicals. To efficiently and accurately deorphanize ORs, we combine the use of a heterologous cell line to express mammalian ORs and a genetically modified biosensor plasmid to measure cAMP production downstream of OR activation in real time. This assay can be used to screen odorants against ORs and vice versa. Positive odorant-receptor interactions from the screens can be subsequently confirmed by testing against various odor concentrations, generating concentration-response curves. Here we used this method to perform a high-throughput screening of an odorous compound against a human OR library expressed in Hana3A cells and confirmed that the positively-responding receptor is the cognate receptor for the compound of interest. We found this high-throughput detection method to be efficient and reliable in assessing OR activation and our data provide an example of its potential use in OR functional studies.
Assuntos
AMP Cíclico/análise , Ensaios de Triagem em Larga Escala/métodos , Receptores Odorantes/análise , Olfato/fisiologia , Técnicas Biossensoriais/métodos , Sistemas Computacionais , AMP Cíclico/genética , AMP Cíclico/metabolismo , Cicloparafinas/análise , Cicloparafinas/metabolismo , Humanos , Medições Luminescentes/métodos , Receptores Odorantes/genética , Receptores Odorantes/metabolismo , Elementos de Resposta , TransfecçãoRESUMO
Receptor transporting protein (RTP) family members, RTP1S and RTP2, are accessory proteins to mammalian odorant receptors (ORs). They are expressed in the olfactory sensory neurons and facilitate OR trafficking to the cell-surface membrane and ligand-induced responses in heterologous cells. We previously identified different domains in RTP1S that are important for different stages of OR trafficking, odorant-mediated responses, and interaction with ORs. However, the exact roles of RTP2 and the significance of the requirement of the seemingly redundant co-expression of the two RTP proteins in vivo have received less attention in the past. Here we attempted to dissect the functional differences between RTP1S and RTP2 using a HEK293T cell-based OR heterologous expression system. When a set of 24 ORs were tested against 28 cognate ligands, unlike RTP1S, which always showed a robust ability to support odorant-mediated responses, RTP2 had little or no effect on OR responses and exhibited a suppressive effect over that of RTP1S for a subset of the ORs tested. RTP1S and RTP2 showed no significant difference in OR ligand selectivity and co-transfection with RTP2 increased the detection threshold for some ORs. A protein-protein interaction analysis showed positive interactions among OR, RTP1S, and RTP2, corroborating the functional linkages among the three molecules. Finally, further cell-surface and permeabilized immunocytochemical studies revealed that OR and the co-expressed RTP1S proteins were retained in the Golgi when co-transfected with RTP2, indicating that RTP1S and RTP2 could play different roles in the OR trafficking process. By examining the functional differentiations between the two RTP family members, we provided a molecular level explanation to the suppressive effect exerted by RTP2, shedding light on the divergent mechanisms underlying the RTP proteins in regulating the functional expression of ORs.
Assuntos
Proteínas de Membrana Transportadoras/genética , Receptores Odorantes/genética , Complexo de Golgi/metabolismo , Células HEK293 , Humanos , Proteínas de Membrana Transportadoras/metabolismo , Neurônios Receptores Olfatórios/metabolismo , Mapas de Interação de Proteínas/genética , Transporte Proteico/genética , Receptores Odorantes/metabolismo , TransfecçãoRESUMO
Covering: up to the end of 2017While suggestions concerning the possible role of metals in olfaction and taste date back 50 years, only recently has it been possible to confirm these proposals with experiments involving individual olfactory receptors (ORs). A detailed discussion of recent experimental results demonstrating the key role of metals in enhancing the response of human and other vertebrate ORs to specific odorants is presented against the backdrop of our knowledge of how the sense of smell functions both at the molecular and whole animal levels. This review emphasizes the role of metals in the detection of low molecular weight thiols, sulfides, and other organosulfur compounds, including those found in strong-smelling animal excretions and plant volatiles, and those used in gas odorization. Alternative theories of olfaction are described, with evidence favoring the modified "shape" theory. The use of quantum mechanical/molecular modeling (QM/MM), site-directed mutagenesis and saturation-transfer-difference (STD) NMR is discussed, providing support for biological studies of mouse and human receptors, MOR244-3 and OR OR2T11, respectively. Copper is bound at the active site of MOR244-3 by cysteine and histidine, while cysteine, histidine and methionine are involved with OR2T11. The binding pockets of these two receptors are found in different locations in the three-dimensional seven transmembrane models. Another recently deorphaned human olfactory receptor, OR2M3, highly selective for a thiol from onions, and a broadly-tuned thiol receptor, OR1A1, are also discussed. Other topics covered include the effects of nanoparticles and heavy metal toxicants on vertebrate and fish ORs, intranasal zinc products and the loss of smell (anosmia).
Assuntos
Receptores Odorantes , Compostos de Enxofre/química , Animais , Humanos , Camundongos , Modelos Moleculares , Estrutura Molecular , Peso Molecular , Odorantes , Neurônios Receptores Olfatórios/fisiologia , VertebradosRESUMO
UNLABELLED: The amygdala serves as emotional center to mediate innate fear behaviors that are reflected through neuronal responses to environmental aversive cues. However, the molecular mechanism underlying the initial neuron responses is poorly understood. In this study, we monitored the innate defensive responses to aversive stimuli of either elevated plus maze or predator odor in juvenile mice and found that glutamatergic neurons were activated in amygdala. Loss of EphB2, a receptor tyrosine kinase expressed in amygdala neurons, suppressed the reactions and led to defects in spine morphogenesis and fear behaviors. We further found a coupling of spinogenesis with these threat cues induced neuron activation in developing amygdala that was controlled by EphB2. A constitutively active form of EphB2 was sufficient to rescue the behavioral and morphological defects caused by ablation of ephrin-B3, a brain-enriched ligand to EphB2. These data suggest that kinase-dependent EphB2 intracellular signaling plays a major role for innate fear responses during the critical developing period, in which spinogenesis in amygdala glutamatergic neurons was involved. SIGNIFICANCE STATEMENT: Generation of innate fear responses to threat as an evolutionally conserved brain feature relies on development of functional neural circuit in amygdala, but the molecular mechanism remains largely unknown. We here identify that EphB2 receptor tyrosine kinase, which is specifically expressed in glutamatergic neurons, is required for the innate fear responses in the neonatal brain. We further reveal that EphB2 mediates coordination of spinogenesis and neuron activation in amygdala during the critical period for the innate fear. EphB2 catalytic activity plays a major role for the behavior upon EphB-ephrin-B3 binding and transnucleus neuronal connections. Our work thus indicates an essential synaptic molecular signaling within amygdala that controls synapse development and helps bring about innate fear emotions in the postnatal developing brain.
Assuntos
Tonsila do Cerebelo/fisiologia , Medo/fisiologia , Glutamatos/metabolismo , Instinto , Neurogênese/fisiologia , Neurônios/fisiologia , Receptor EphB2/metabolismo , Envelhecimento/fisiologia , Animais , Mecanismos de Defesa , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Transdução de Sinais/fisiologiaRESUMO
Mammalian survival depends on ultrasensitive olfactory detection of volatile sulfur compounds, since these compounds can signal the presence of rancid food, O2 depleted atmospheres, and predators (through carnivore excretions). Skunks exploit this sensitivity with their noxious spray. In commerce, natural and liquefied gases are odorized with t-BuSH and EtSH, respectively, as warnings. The 100-million-fold difference in olfactory perception between structurally similar EtSH and EtOH has long puzzled those studying olfaction. Mammals detect thiols and other odorants using odorant receptors (ORs), members of the family of seven transmembrane G-protein-coupled receptors (GPCRs). Understanding the regulator cofactors and response of ORs is particularly challenging due to the lack of X-ray structural models. Here, we combine computational modeling and site-directed mutagenesis with saturation transfer difference (STD) NMR spectroscopy and measurements of the receptor response profiles. We find that human thiol receptor OR2T11 responds specifically to gas odorants t-BuSH and EtSH requiring ionic copper for its robust activation and that this role of copper is mimicked by ionic and nanoparticulate silver. While copper is both an essential nutrient for life and, in excess, a hallmark of various pathologies and neurodegenerative diseases, its involvement in human olfaction has not been previously demonstrated. When screened against a series of alcohols, thiols, sulfides, and metal-coordinating ligands, OR2T11 responds with enhancement by copper to the mouse semiochemical CH3SCH2SH and derivatives, to four-membered cyclic sulfide thietane and to one- to four-carbon straight- and branched-chain and five-carbon branched-chain thiols but not to longer chain thiols, suggesting compact receptor dimensions. Alcohols are unreactive.
RESUMO
BitterX is an open-access tool aimed at providing a platform for identifying human bitter taste receptors, TAS2Rs, for small molecules. It predicts TAS2Rs from the molecular structures of arbitrary chemicals by integrating two individual functionalities: bitterant verification and TAS2R recognition. Using BitterX, several novel bitterants and their receptors were predicted and experimentally validated in the study. Therefore, BitterX may be an effective method for deciphering bitter taste coding and could be a useful tool for both basic bitter research in academia and new bitterant discoveries in the industry.
Assuntos
Receptores Acoplados a Proteínas G/metabolismo , Bibliotecas de Moléculas Pequenas/metabolismo , Paladar/genética , Algoritmos , Bases de Dados de Compostos Químicos , Células HEK293 , Humanos , Receptores Acoplados a Proteínas G/química , SoftwareRESUMO
Innate emotion response to environmental stimuli is a fundamental brain function that is controlled by specific neural circuits. Dysfunction of early emotional circuits may lead to neurodevelopmental disorders such as autism and schizophrenia. However, how the functional circuits are formed to prime initial emotional behaviours remain elusive. We reveal here using gene-targeted mutations an essential role for ephrin-B3 ligand-like activity in the development of innate fear in the neonatal brain. We further demonstrate that ephrin-B3 controls axon targeting and coordinates spinogenesis and neuronal activity within the amygdala. The morphological and behavioural abnormalities in ephrin-B3 mutant mice are rescued by conditional knock-in of wild-type ephrin-B3 during the critical period when axon targeting and fear responses are initiated. Our results thus define a key axonal molecule that participates in the wiring of amygdala circuits and helps bring about fear emotion during the important adolescence period.
Assuntos
Tonsila do Cerebelo/metabolismo , Axônios/metabolismo , Efrina-B3/metabolismo , Medo/fisiologia , Instinto , Neurogênese , Animais , Núcleo Celular/metabolismo , Efrina-B3/genética , Camundongos , Mutação/genética , Sinapses/metabolismo , Fatores de TempoAssuntos
Modelos Biológicos , Odorantes , Receptores Odorantes/metabolismo , Olfato/fisiologia , Animais , HumanosRESUMO
The vibrational theory of olfaction assumes that electron transfer occurs across odorants at the active sites of odorant receptors (ORs), serving as a sensitive measure of odorant vibrational frequencies, ultimately leading to olfactory perception. A previous study reported that human subjects differentiated hydrogen/deuterium isotopomers (isomers with isotopic atoms) of the musk compound cyclopentadecanone as evidence supporting the theory. Here, we find no evidence for such differentiation at the molecular level. In fact, we find that the human musk-recognizing receptor, OR5AN1, identified using a heterologous OR expression system and robustly responding to cyclopentadecanone and muscone, fails to distinguish isotopomers of these compounds in vitro. Furthermore, the mouse (methylthio)methanethiol-recognizing receptor, MOR244-3, as well as other selected human and mouse ORs, responded similarly to normal, deuterated, and (13)C isotopomers of their respective ligands, paralleling our results with the musk receptor OR5AN1. These findings suggest that the proposed vibration theory does not apply to the human musk receptor OR5AN1, mouse thiol receptor MOR244-3, or other ORs examined. Also, contrary to the vibration theory predictions, muscone-d30 lacks the 1,380- to 1,550-cm(-1) IR bands claimed to be essential for musk odor. Furthermore, our theoretical analysis shows that the proposed electron transfer mechanism of the vibrational frequencies of odorants could be easily suppressed by quantum effects of nonodorant molecular vibrational modes. These and other concerns about electron transfer at ORs, together with our extensive experimental data, argue against the plausibility of the vibration theory.
Assuntos
Modelos Biológicos , Odorantes , Receptores Odorantes/metabolismo , Olfato/fisiologia , Animais , Isótopos de Carbono , Cicloparafinas/química , Deutério , Transporte de Elétrons , Ácidos Graxos Monoinsaturados/química , Células HEK293 , Humanos , Isomerismo , Camundongos , VibraçãoRESUMO
Understanding structure/function relationships of olfactory receptors is challenging due to the lack of x-ray structural models. Here, we introduce a QM/MM model of the mouse olfactory receptor MOR244-3, responsive to organosulfur odorants such as (methylthio)methanethiol. The binding site consists of a copper ion bound to the heteroatoms of amino-acid residues H105, C109, and N202. The model is consistent with site-directed mutagenesis experiments and biochemical measurements of the receptor activation, and thus provides a valuable framework for further studies of the sense of smell at the molecular level.
Assuntos
Receptores Odorantes/química , Animais , Sítios de Ligação , Simulação por Computador , Cobre/química , Humanos , Íons/química , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Estrutura Secundária de Proteína , Teoria Quântica , Receptor Muscarínico M2/química , Receptores Odorantes/genética , Alinhamento de Sequência , Relação Estrutura-Atividade , Água/químicaRESUMO
Humans have ~400 intact odorant receptors, but each individual has a unique set of genetic variations that lead to variation in olfactory perception. We used a heterologous assay to determine how often genetic polymorphisms in odorant receptors alter receptor function. We identified agonists for 18 odorant receptors and found that 63% of the odorant receptors we examined had polymorphisms that altered in vitro function. On average, two individuals have functional differences at over 30% of their odorant receptor alleles. To show that these in vitro results are relevant to olfactory perception, we verified that variations in OR10G4 genotype explain over 15% of the observed variation in perceived intensity and over 10% of the observed variation in perceived valence for the high-affinity in vitro agonist guaiacol but do not explain phenotype variation for the lower-affinity agonists vanillin and ethyl vanillin.
Assuntos
Variação Genética , Percepção Olfatória/genética , Receptores Odorantes/metabolismo , Olfato/fisiologia , Adulto , Idoso , Relação Dose-Resposta a Droga , Feminino , Frequência do Gene , Genótipo , Guaiacol/farmacologia , Humanos , Modelos Lineares , Masculino , Pessoa de Meia-Idade , Odorantes , Polimorfismo de Nucleotídeo Único , Psicofísica , Receptores Odorantes/genética , Adulto JovemRESUMO
Odorant receptor (OR) proteins are retained in the endoplasmic reticulum when heterologously expressed in cultured cells of non-olfactory origins. RTP1S is an accessory protein to mammalian ORs and facilitates their trafficking to the cell-surface membrane and ligand-induced responses in heterologous cells. The mechanism by which RTP1S promotes the functional expression of ORs remains poorly understood. To obtain a better understanding of the role(s) of RTP1S, we performed a series of structure-function analyses of RTP1S in HEK293T cells. By constructing RTP1S deletion and chimera series and subsequently introducing single-site mutations into the protein, we found the N terminus of RTP1S is important for the endoplasmic reticulum exit of ORs and that a middle region of RTP1S is important for OR trafficking from the Golgi to the membrane. Using sucrose gradient centrifugation, we found that the localization of RTP1S to the lipid raft microdomain is critical to the activation of ORs. Finally, in a protein-protein interaction analysis, we determined that the C terminus of RTP1S may be interacting with ORs. These findings provide new insights into the distinct roles of RTP1S in OR translocation and activation.
Assuntos
Proteínas de Membrana Transportadoras/química , Receptores Odorantes/metabolismo , Citometria de Fluxo/métodos , Células HEK293 , Humanos , Imuno-Histoquímica/métodos , Ligantes , Microdomínios da Membrana/metabolismo , Proteínas de Membrana Transportadoras/fisiologia , Modelos Biológicos , Plasmídeos/metabolismo , Estrutura Terciária de Proteína , Transporte Proteico , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Odorant receptors (ORs) in olfactory sensory neurons (OSNs) mediate detection of volatile odorants. Divalent sulfur compounds, such as thiols and thioethers, are extremely potent odorants. We identify a mouse OR, MOR244-3, robustly responding to (methylthio)methanethiol (MeSCH(2)SH; MTMT) in heterologous cells. Found specifically in male mouse urine, strong-smelling MTMT [human threshold 100 parts per billion (ppb)] is a semiochemical that attracts female mice. Nonadjacent thiol and thioether groups in MTMT suggest involvement of a chelated metal complex in MOR244-3 activation. Metal ion involvement in thiol-OR interactions was previously proposed, but whether these ions change thiol-mediated OR activation remained unknown. We show that copper ion among all metal ions tested is required for robust activation of MOR244-3 toward ppb levels of MTMT, structurally related sulfur compounds, and other metal-coordinating odorants (e.g., strong-smelling trans-cyclooctene) among >125 compounds tested. Copper chelator (tetraethylenepentamine, TEPA) addition abolishes the response of MOR244-3 to MTMT. Histidine 105, located in the third transmembrane domain near the extracellular side, is proposed to serve as a copper-coordinating residue mediating interaction with the MTMT-copper complex. Electrophysiological recordings of the OSNs in the septal organ, abundantly expressing MOR244-3, revealed neurons responding to MTMT. Addition of copper ion and chelator TEPA respectively enhanced and reduced the response of some MTMT-responding neurons, demonstrating the physiological relevance of copper ion in olfaction. In a behavioral context, an olfactory discrimination assay showed that mice injected with TEPA failed to discriminate MTMT. This report establishes the role of metal ions in mammalian odor detection by ORs.
Assuntos
Cobre/fisiologia , Odorantes , Neurônios Receptores Olfatórios/metabolismo , Receptores Odorantes/química , Atrativos Sexuais/metabolismo , Compostos de Sulfidrila/metabolismo , Sulfetos/metabolismo , Sequência de Aminoácidos , Animais , Cátions/farmacologia , Quelantes/farmacologia , AMP Cíclico/análise , Relação Dose-Resposta a Droga , Etilenodiaminas/farmacologia , Feminino , Histidina/química , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Dados de Sequência Molecular , Técnicas de Patch-Clamp , Conformação Proteica , Estrutura Terciária de Proteína , Receptores Odorantes/genética , Receptores Odorantes/fisiologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , Especificidade por Substrato , Compostos de Enxofre/metabolismoRESUMO
SUMMARY: ODORactor is an open access web server aimed at providing a platform for identifying odorant receptors (ORs) for small molecules and for browsing existing OR-ligand pairs. It enables the prediction of ORs from the molecular structures of arbitrary chemicals by integrating two individual functionalities: odorant verification and OR recognition. The prediction of the ORs for several odorants was experimentally validated in the study. In addition, ODORactor features a comprehensive repertoire of olfactory information that has been manually curated from literature. Therefore, ODORactor may provide an effective way to decipher olfactory coding and could be a useful server tool for both basic olfaction research in academia and for odorant discovery in industry. AVAILABILITY: Freely available at http://mdl.shsmu.edu.cn/ODORactor CONTACT: jian.zhang@sjtu.edu.cn SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Receptores Odorantes/química , Software , Animais , Humanos , Internet , Ligantes , Camundongos , Odorantes , OlfatoRESUMO
The treatment outcome of acute lymphoblastic leukemia (ALL) has improved steadily over the last 50 years. However, the cure rates are unlikely to be raised further with current therapies. Since increasing the dosage of chemotherapeutic agents could also elevate toxicity, a solution to how one could achieve maximum therapeutic effect with the minimum dosage possible is imminent. One possibility is the employment of combination drug therapies. Arsenic trioxide (ATO) is a widely used drug for acute promyelocytic leukemia (APL). Its combination with other drugs presented therapeutic activities in malignant cancers other than APL. Considering the fact that ATO induces mitotic arrest prior to apoptosis induction, we attempted to investigate the potential anti-cancer effects of ATO in combination with the microtubule-stabilizing agent, paclitaxel (PTX), using malignant lymphocytes as in vitro models. Three malignant lymphocytic cell lines and primary cells were treated with ATO and/or PTX. Using the Chou-Talalay analysis for evaluation of combined effect of ATO and PTX, we found a synergistic effect of the two drugs in the inhibition of cell growth. We also found that the combination of ATO and PTX at low concentrations synergistically induced mitotic arrest followed by apoptosis in malignant lymphocytes, which increased phosphorylated cyclin-dependent kinase 1 (Cdk1) on Thr(161) and promoted the dysregulated activation of Cdk1. The ATO/PTX combination also significantly enhanced the activation of spindle checkpoint by inducing the formation of the inhibitory checkpoint complex BubR1/Cdc20. Our study provided the first in vitro demonstration that low concentrations of ATO and PTX synergistically induce mitotic arrest in malignant lymphocytes.
Assuntos
Antineoplásicos/farmacologia , Arsenicais/farmacologia , Mitose/efeitos dos fármacos , Óxidos/farmacologia , Paclitaxel/farmacologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Moduladores de Tubulina/farmacologia , Apoptose , Trióxido de Arsênio , Proteína Quinase CDC2/metabolismo , Células Cultivadas , Sinergismo Farmacológico , Humanos , Células JurkatRESUMO
Odorant receptors are among the fastest evolving genes in animals. However, little is known about the functional changes of individual odorant receptors during evolution. We have recently demonstrated a link between the in vitro function of a human odorant receptor, OR7D4, and in vivo olfactory perception of 2 steroidal ligands--androstenone and androstadienone--chemicals that are shown to affect physiological responses in humans. In this study, we analyzed the in vitro function of OR7D4 in primate evolution. Orthologs of OR7D4 were cloned from different primate species. Ancestral reconstruction allowed us to reconstitute additional putative OR7D4 orthologs in hypothetical ancestral species. Functional analysis of these orthologs showed an extremely diverse range of OR7D4 responses to the ligands in various primate species. Functional analysis of the nonsynonymous changes in the Old World Monkey and Great Ape lineages revealed a number of sites causing increases or decreases in sensitivity. We found that the majority of the functionally important residues in OR7D4 were not predicted by the maximum likelihood analysis detecting positive Darwinian selection.