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.

项目概要味觉家族 1 受体（T1R，形成甜味和鲜味受体）和味觉家族 2 受体（T2R，形成苦味受体）是在味蕾中表达的重要 G 蛋白偶联受体 (GPCR) T1R 和 T2R 在全身组织（包括舌头）中也发挥着重要的化学感应作用。我们和其他人将 T1R 和 T2R 描述为新的参与者。他们检测细菌代谢物以刺激快速的先天免疫反应。虽然目前的筛选方法已经揭示了许多有关激活特定 T1R 的特定激动剂的信息和 T2R，了解复杂混合物（例如植物提取物或条件化混合物）对 T1R 和 T2R 的激活现有的筛选方法目前非常困难，主要集中在钙信号传导上。许多化合物独立于培养细胞中的味觉受体激活钙信号，并且常用的表达内源性 T2R 的 HEK293 细胞也有以钙为重点的筛选方法。可能错过了“偏向激动剂”，即不激活 T2R G 蛋白信号传导但激活许多 GPCR 都有这种情况的报道，但尚未有人筛选出有偏向的激动剂。这是 T1R 和 T2R 生物学研究中的一个关键空白，本文将予以解决。为了克服基于钙的检测的局限性并揭示对 T2R-arrestin 信号传导的新见解，我们将采用基于荧光的三方 GFP 测定法（称为 TRIO 测定法），该测定法可直接这通过视紫红质抑制蛋白与激活的受体结合来可视化异源表达的受体激活。检测受到复杂混合物或单一比较化合物的脱靶效应的限制要小得多它也适用于高通量酶标仪仪器，该测定速度更快 (1-2)。小时）比其他依赖转录作为读数的 GPCR 测定（12-24 小时）我们使用了这种测定。研究前期研究中蛋白酶激活受体和肾上腺素受体的激活和抑制。数据表明该测定与 T2R 配合良好，我们希望 T1R 和 T2R TRIO 测定能够揭示这一点。新的配体-T1R/T2R 相互作用，可能有助于去孤儿化剩余的孤儿 T2R 亚型。在本提案中，目标 1 将重点关注使用已知的苦味和甜味来验证这种光学测定目标 2 将使用该测定来筛选针对常见病原菌和化合物的味觉受体。真菌来表征哪些受体被哪些病原体激活将是新的临床相关数据。揭示了，本研究中验证的表达结构将成为味觉受体的有用工具所有表达载体将在首次发表后通过 Addgene 广泛提供。研究对于免疫研究以及分子感官营养和味觉具有重要用途研究。