Development and validation of novel optical methods for direct screening of taste receptor activation

直接筛选味觉受体激活的新型光学方法的开发和验证

基本信息

批准号：
10593556
负责人：
Robert J. Lee
金额：
$ 24.38万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-12-01 至 2024-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10593556
关键词：
Address Adrenergic Receptor Agonist Amino Acids Arrestins Aspergillus flavus Aspergillus fumigatus Bacteria Binding Binding Proteins Biological Assay Biology Calcium Calcium Channel Calcium Signaling Cells Clinical Complex Mixtures Couples Cultured Cells Cyclic AMP Data Development Epithelial Cells Epithelium Exhibits Family Fluo 4 Fluorescence Food Future G-Protein-Coupled Receptors GTP-Binding Proteins Genetic Transcription Glutaminase Glutamine Heterotrimeric GTP-Binding Proteins High Pressure Liquid Chromatography Immune Immunologic Receptors Inhalation Innate Immune Response Ligands Mediating Methicillin Methicillin Resistance Methods Molecular Natural Immunity Nutritional Study Optical Methods Optics Orphan Pathway interactions Peptide Hydrolases Phospholipase C Phospholipases A Phosphotransferases Plant Extracts Play Potassium Channel Protein Isoforms Pseudomonas aeruginosa Publications Publishing Quinine Reader Receptor Activation Reporting Research Role Sensory Signal Pathway Signal Transduction Signaling Protein Staphylococcus aureus Taste Buds Taste Perception Testing Tissues Tongue Validation Vegetables Visualization Work airway epithelium arm calcium indicator clinically relevant expression vector fungus high throughput screening improved insight instrumentation novel nutrition pathogen pathogenic bacteria pathogenic fungus preservation prevent rat Gnat3 protein receptor respiratory smooth muscle response screening sweet receptor sweet taste perception taste transduction tool voltage

项目摘要

PROJECT SUMMARY Taste family 1 receptors (T1Rs, forming sweet and umami receptors) and taste family 2 receptors (T2Rs, forming bitter receptors) are important G-protein couple receptors (GPCRs) expressed in taste buds of the tongue. T1Rs and T2Rs also play important chemosensory roles in tissues all over the body, including the airway epithelium and in immune cells. We and other have characterized T1Rs and T2Rs as novel players in innate immunity. They detect bacterial metabolites to stimulate rapid innate immune responses. While current screening methods have revealed a lot about specific agonists that activate specific T1Rs and T2Rs, understanding T1R and T2R activation by complex mixtures (e.g., plant extracts or conditioned media from bacteria) is currently very difficult. Existing screening methods are focused on calcium signaling as a read-out. Many compounds activate calcium signals independently of taste receptors in cultured cells, and the HEK293 cells commonly used express endogenous T2Rs. Calcium-focused screening methods have also likely missed “biased agonists,” that is, agonists which do not activate T2R G protein signaling but activate arrestin signaling. This has been reported for many GPCRs, but no one has yet screened for biased agonists of taste receptors. This is a critical gap in the studies of T1R and T2R biology that will be addressed here. To overcome limitations of calcium-based assays and reveal new insights into T2R-arrestin signaling, we will adapt a fluorescence-based tripartite GFP-based assay (known as the TRIO assay) that directly visualizes heterologously-expressed receptor activation through arrestin binding to the activated receptor. This assay is much less limited by off-target effects of complex mixtures or single compounds compared with current methods. It is also adaptable to high throughput plate-reader instrumentation. This assay is faster (1-2 hrs) than other GPCR assays which rely on transcription as a readout (12-24 hrs). We have utilized this assay to study activation and inhibition of protease-activated and adrenergic receptors in prior studies. Preliminary data suggest this assay works well with T2Rs. We hypothesize that T1R and T2R TRIO assays will reveal novel ligand-T1R/T2R interactions and may be useful to de-orphanize the remaining orphan T2R isoforms. In this proposal, Aim 1 will focus on validation of this optical assay using known bitter and sweet compounds. Aim 2 will use this assay to screen taste receptors against common pathogenic bacteria and fungi to characterize which receptors are activated by which pathogens. New clinically relevant data will be revealed, and the expression constructs validated in this study will become useful tools for taste receptor research. All expression vectors will be made widely available via Addgene after initial publication. This research has important utility for both immune research as well as molecular sensory nutrition and taste research.

项目摘要味道家族1受体（T1RS，形成甜和鲜味受体）和味道家族2受体（T2R，形成苦受体）是重要的G蛋白夫妇受体（GPCR），该受体在味蕾中表达舌头。 T1RS和T2RS在整个组织中也起着重要的化学感应作用，包括气道上皮和免疫小球。我们和其他人将T1R和T2Rs描述为新颖的球员先天免疫。他们检测细菌代谢物以刺激先天性免疫反应。虽然当前的筛选方法已经揭示了激活特定T1RS的特定激动剂的很多和T2RS，通过复杂混合物了解T1R和T2R激活（例如植物提取物或条件来自细菌的媒体）目前非常困难。现有的筛选方法集中于钙信号传导读出。许多化合物都独立于培养细胞中的味觉受体激活钙信号，并且 HEK293细胞通常使用表达内源性T2R。以钙为中心的筛查方法也具有可能错过了“偏见激动剂”，即不会激活T2R G蛋白信号但激活的激动剂逮捕蛋白信号传导。据报道，这是许多GPCR的报道，但还没有人筛选有偏见的激动剂味道受体。这是将在此处解决的T1R和T2R生物学研究的关键差距。为了克服基于钙的评估的局限性，并揭示了对T2R-arrestin信号传导的新见解，我们将调整基于荧光的三方GFP测定法（称为三重奏测定），直接通过阻止蛋白与活化受体结合，可视化异源表达的受体激活。这相比当前方法。它也适用于高通量板读取器仪器。该测定速度更快（1-2 HRS）比其他依赖转录作为读数的GPCR分析（12-24小时）。我们已经使用了此测定法在先前的研究中研究蛋白酶激活和肾上腺素受体的激活和抑制。初步的数据表明，该测定法与T2RS效果很好。我们假设T1R和T2R三重奏测定将揭示新型的配体-T1R/T2R相互作用，可能有助于脱键化其余的孤儿T2R同工型。在此提案中，AIM 1将专注于使用已知苦和甜的验证该光学测定化合物。 AIM 2将使用此测定法对常见的致病细菌进行筛选味道受体和真菌表征哪些受体被哪些病原体激活。新的临床相关数据将是揭示了，本研究中验证的表达结构将成为味觉接收器的有用工具研究。首次出版后，所有表达式向量将通过addgene广泛使用。这研究对于免疫研究以及分子感觉营养和品味都有重要的效用研究。