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

• 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
• 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

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.

项目摘要 赤潮严重破坏海岸带生态系统，引发一系列环境疾病， 居住在海岸附近或食用受红潮污染的海产的人士。尽管他们的生态和 环境意义，对赤潮发生机理的研究有限。 质膜G蛋白偶联受体（GPCRs）广泛参与真核生物的多细胞信号转导 转导，但其在单细胞真核生物中的作用尚未得到充分探索。最近的研究 暗示原始GPCR参与甲藻的生物发光途径，包括 多边沟甲藻L. polyedra是一种多产的单细胞甲藻，与赤潮有关， 对波浪湍流和捕食者行为高度敏感。Gaq型异源三聚体G蛋白， GPCR信号传导中的信号通路，已被广泛报道其与 机械化学换能器初步的生物信息学分析表明，L. polyedra GPCR是 在生物发光的途径中。对前5种受体候选物（RC 1至RC 5）进行了以下处理： 反义DNA寡核苷酸敲除实验。击倒表明RC 3是一个有前途的 参与生物发光信号的候选人。根据我们的初步数据，R21项目将进一步 探讨RC 3在甲藻L. polyedra生物发光，从确认其剪切力开始- 敏感性，分子上确定其在生物发光信号传导中的作用， 受体和相关信号分子。该项目的三个具体目标包括：目标1，RT-qPCR 将用于评估RC 3及其效应物在信号通路中的mRNA水平。CRISPR/Cas9 敲除策略也将用于验证RC 3在剪切力引起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制备系统中异源表达RC 3，然后使用 基于单溴联苯胺的荧光光谱学来研究其对剪切力的构象响应， copepodamide脂质。在目的3中，我们将确定RC 3相关的生物发光信号通路是否是Gaq-1。 依赖性，使用敲除和敲除实验沿着体外测量，包括 GTP酶-GloTM测定和基于[3 H]GDP的GDP释放测定。R21项目数据将决定 甲藻衍生的GPCR-RC 3与剪切力诱导的生物发光相关。该项目的 该实验的完成为全面研究GPCR信号转导的分子机制奠定了基础 在赤潮中的作用，对控制或调节赤潮的发生具有深远的意义。