Towards molecular mechanisms of invertebrate Gustatory Receptors

无脊椎动物味觉受体的分子机制

基本信息

批准号：
10064623
负责人：
RACHELLE GAUDET
金额：
$ 19.82万
依托单位：
BRANDEIS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-01-01 至 2022-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10064623
关键词：
Behavior Binding Biochemical Biochemistry Blood Buffers Carbon Cells Chemicals Collaborations Complex Cryoelectron Microscopy Crystallography Cues Culicidae Data Dengue Diet Disease Disease Vectors Drosophila melanogaster Family Family member Fructose Future Goals Homo Human Insecta Invertebrates Ion Channel Gating Ligands Malaria Membrane Membrane Proteins Modality Molecular Nramp protein Nutrient Orthologous Gene Physiological Play Positioning Attribute Preparation Property Proteins Protocols documentation Research Personnel Role Sensory Sensory Receptors Sequence Analysis Smell Perception Stimulus Structure System Taste Perception Testing Ticks Variant West Nile virus Work ZIKA chikungunya comparative disorder control extreme temperature feeding fly human disease insect disease vector member olfactory receptor receptor receptor function receptor structure function sensor success sugar vector control

项目摘要

PROJECT SUMMARY Invertebrate Gustatory Receptors (GRs) are a large and evolutionarily diverse family of sensory receptors known to play important roles in invertebrate taste, smell and thermotransduction. Given the importance of these sensory modalities in host-seeking behavior in important humand disease vectors like mosquitoes, GR family members serve as potentially powerful targets for vector control agents. However, little is known about GR structure and function. We propose a physiological and biochemical analysis of members of two GR subfamilies: Gr43a and Gr28bD. These initial studies will serve as a precursor for a subsequent R01 to carry out structural and functional analyses of these GRs. We propose to achieve these goals in two aims: Aim #1: Identify and physiologically characterize multiple orthologs of Gr43a and Gr28bD. Unlike most GRs, Gr43a and Gr28bD orthologs can be functionally characterized in heterologous cells. In aim 1.a., we will express orthologs of these GRs from additional insect species, including disease vectors and extremophiles, in heterologous cells and characterize their physiological properties. This will enable a comparative analysis of sequence and function among each receptor class. Aim #2: Biochemically characterize multiple Gr43a and Gr28bD orthologs. We find Gr43a and Gr28bD orthologs can be partially purified from heterologous cells. In aim 2, we will expand this approach to incorporate additional orthologs characterized in aim 1 and optimize our purification protocol and explore key properties including oligomeric state and thermal stability in various membrane mimics. This will provide important biochemical information about GR complexes and identify orthologs best suited for subsequent structural analysis. The physiological characterization of multiple Gr43a and Gr28bD orthologs will enable direct examination of evolutionary variation and conservation in GR family function. The expression and purification of multiple family members will provide multiple candidates for biochemistry and structural determination, maximizing the likelihood of success of subsequent GR structural determinations.

项目摘要无脊椎动物味觉受体（GRS）是一个大而进化的感觉受体家族已知会在无脊椎动物的味道，气味和热转移中扮演重要角色。考虑到重要性在重要的性性疾病媒介（如蚊子，GR）中，这些在寻求宿主行为的感觉方式家庭成员是对向量控制剂的潜在强大目标。但是，对 GR结构和功能。我们提出了两个GR成员的生理和生化分析亚家族：GR43A和GR28BD。这些初步研究将作为随后的R01的前体这些GRS的结构和功能分析。我们建议以两个目的实现这些目标： AIM＃1：识别和生理表征GR43A和GR28BD的多个直系同源物。与大多数GRS不同，GR43A和GR28BD直系同源物可以在异源细胞中功能表征。目标 1.A.，我们将从其他昆虫物种中表达这些GRS的直系同源物，包括疾病媒介和在异源细胞中的极端细胞并表征其生理特性。这将使每个受体类别之间序列和功能的比较分析。 AIM＃2：生化表征多个GR43A和GR28BD直系同源物。我们找到了Gr43a和 GR28BD直系同源物可以从异源细胞中部分纯化。在AIM 2中，我们将将这种方法扩展到合并AIM 1中特征的其他直系同源物并优化我们的纯化协议并探索密钥各种膜模拟物中的特性，包括寡聚状态和热稳定性。这将提供有关GR复合物的重要生化信息，并确定最适合随后的直系同源结构分析。多个GR43A和GR28BD直系同源物的生理表征将直接检查 GR家族功能中的进化变化和保护。多重的表达和纯化家庭成员将为生物化学和结构决心提供多个候选人，最大化随后的GR结构确定成功的可能性。