The Role of G Protein-coupled Receptors in Red Tide Dinoflagellate Bioluminescence

G 蛋白偶联受体在赤潮甲藻生物发光中的作用

基本信息

批准号：
10708533
负责人：
Libin Ye
金额：
$ 40.68万
依托单位：
UNIVERSITY OF SOUTH FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-08-23 至 2025-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10708533
关键词：
Affect Agonist Algae Anabolism Angiotensins Antisense DNA Behavior Binding Bioinformatics Biological Biological Assay Bioluminescence Biophysics Blood Blood Pressure Bradykinin CRISPR/Cas technology Cell membrane Cells Codon Nucleotides Consumption Copepoda Cysteine Data Diarrhea Dinophyceae Disease Eating Ecosystem Endothelial Cells Equilibrium Eukaryota Event Fisheries Fishes Fluorescence Microscopy Fluorescence Resonance Energy Transfer Foundations G Protein-Coupled Receptor Signaling G-Protein-Coupled Receptors GPR68 gene GTP-Binding Proteins Genes Genomics Guanosine Triphosphate Phosphohydrolases Health Heterotrimeric GTP-Binding Proteins Human Hydrolysis In Vitro Integral Membrane Protein Knock-out Ligands Link Lipids Liquid substance MAS1 gene Measurement Mediating Membrane Memory Loss Messenger RNA Molecular Molecular Conformation Monitor NMR Spectroscopy Nausea Nucleotides Oligonucleotides Organism Outcome Pathway interactions Persons Pharmaceutical Preparations Pichia Plasma Predatory Behavior Preparation Protein Inhibition Proteins Receptor Activation Reporting Research Respiratory Paralysis Rhodopsin Role Seafood Series Shellfish Signal Pathway Signal Transduction Spectrum Analysis Stress System Tertiary Protein Structure Toxin Transducers Transmembrane Domain Yeasts blood pressure regulation contaminated seafood experimental study fluidity insight knock-down mutant pressure receptor red tide response shear stress time use transcriptome transcriptomics

项目摘要

Project Summary Red tide blooms significantly cause coastal ecosystem damage and a series of environmental diseases on people who live nearby the coast or eat the red tide contaminated seafoods. Despite their ecological and environmental significance, research in mechanistically understanding the red tide bloom occurrence is limited. Plasma membrane G protein-coupled receptor (GPCRs) are widely implicated in multi-cellular eukaryotic signal transductions, but their roles in the unicellular eukaryotic organism have not been well-explored. Recent studies implied the involvements of primitive GPCRs in the bioluminescent pathway of dinoflagellates, including Lingulodinium polyedra. L. polyedra is a prolific single-celled dinoflagellate implicated in red tide events and highly sensitive to wave turbulence and predator behavior. Gaq-type heterotrimeric G proteins, one of major signal pathways in the GPCR signaling, have been extensively reported for their associations with mechanochemical transducer. Our preliminary bioinformatic analysis indicate a set of L. polyedra GPCRs are implied in the bioluminescent pathway. The top 5 receptor candidates (RC1 through RC5) were subjected to anti-sense DNA oligonucleotide knockdown experiments. The knockdowns indicate that RC3 is a promising candidate involved in the bioluminescent signaling. Based on our preliminary data, this R21 project will further explore the role of RC3 in dinoflagellate L. polyedra bioluminescence, starting from confirmation its shear force- sensitivity, molecularly determining its role in bioluminescent signaling and biophysically characterizing the receptor and related signaling partners. The project’s three specific aims include the following: Aim 1, RT-qPCR will be used for evaluating the mRNA levels of RC3 and its effectors in the signaling pathway. CRISPR/Cas9 knockout strategy will also be used for validating the role of RC3 in shear-force elicited bioluminescence signaling. In Aim 2, we will first heterogeneously express RC3 in a yeast-based GPCR preparation system and then use monobromobiamine-based florescent spectroscopy to study its conformational responses to shear-forces and copepodamide lipids. In Aim 3, we will determine whether RC3 related bioluminescence signal pathway is Gaq- dependent, using both knockdown and knockout experiments along with in vitro measurements, including GTPase-GloTM assay and [3H]GDP based GDP release assay. The R21 project data will determine if the dinoflagellate derived GPCR—RC3 is associated with shear force-induced bioluminescence. The project’s completion will establish a priming foundation to completely study the molecular mechanism of GPCR signaling in the red tide blooms, with the far-reaching significance of controlling or modulating bloom occurrences.

项目概要赤潮水华严重造成沿海生态系统破坏和一系列环境病害居住在海岸附近的人们或食用了赤潮污染的海鲜，尽管它们的生态和安全性较差。环境意义，从机制上理解赤潮水华发生的研究是有限的。质膜 G 蛋白偶联受体 (GPCR) 广泛涉及多细胞真核信号转导，但它们在单细胞真核生物中的作用尚未得到充分探索。暗示原始 GPCR 参与甲藻的生物发光途径，包括 Lingulodinium polyedra 是一种与赤潮事件有关的多产单细胞甲藻。对波浪湍流和捕食者行为高度敏感，Gaq 型异三聚体 G 蛋白是主要的蛋白之一。 GPCR 信号通路中的信号通路主要因其与我们的初步生物信息分析表明一组 L. polyedra GPCR 是生物发光途径中暗示的前 5 个受体候选者（RC1 至 RC5）受到影响。反义 DNA 寡核苷酸敲低实验表明 RC3 是一种有前途的药物。根据我们的初步数据，该 R21 项目将进一步研究参与生物发光信号的候选者。探讨RC3在甲藻L.polyedra生物发光中的作用，从确认其剪切力开始- 敏感性，从分子角度确定其在生物发光信号传导中的作用以及生物物理特征该项目的三个具体目标包括：目标 1，RT-qPCR。将用于评估 RC3 及其在信号通路中的效应子的 mRNA 水平。敲除策略还将用于验证 RC3 在剪切力引发的生物发光信号传导中的作用。在目标 2 中，我们将首先在基于酵母的 GPCR 制备系统中异质表达 RC3，然后使用基于一溴二胺的荧光光谱研究其对剪切力的构象响应在目标3中，我们将确定RC3相关的生物发光信号通路是否为Gaq-。依赖性，使用击倒和敲除实验以及体外测量，包括 R21 项目数据将确定 GTPase-GloTM 测定和基于 [3H]GDP 的 GDP 释放测定。甲藻衍生的 GPCR-RC3 与剪切力诱导的生物发光有关。该项目的完成将为全面研究GPCR信号传导的分子机制奠定基础在赤潮水华中，具有控制或调节水华发生的深远意义。