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 红潮血会显着造成沿海生态系统损害，并在 居住在海岸附近或吃红潮污染的海鲜的人。尽管他们的生态和 环境意义，机械理解红潮开花的研究是有限的。 质膜G蛋白偶联受体（GPCR）在多细胞真核信号中广泛实现 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。最近的研究 暗示原始GPCR参与鞭毛鞭毛的生物发光途径，包括 lingulodinium polyedra。 L. Polyedra是一种多产的单细胞鞭毛藻，与红潮发生和 GAQ型异三聚体G蛋白，主要之一 GPCR信号中的信号途径已广泛报道了其与 机械化学换能器。我们的初步生物信息学分析表明，一组Polyedra GPCR是 在生物发光途径中实施。候选前5名受体（RC1至RC5）进行 反义DNA寡核苷酸敲低实验。敲低表明RC3是一个承诺 参与生物发光信号传导的候选者。根据我们的初步数据，此R21项目将进一步 探索RC3在鞭毛藻中的作用，从确认其剪切力开始 敏感性，分子确定其在生物发光信号传导中的作用，并以生物物理为特征 接收器和相关信号伙伴。该项目的三个具体目的包括以下内容：AIM 1，RT-QPCR 将用于评估RC3的mRNA水平及其在信号通路中的影响。 CRISPR/CAS9 敲除策略还将用于验证RC3在剪切力引起的生物发光信号传导中的作用。 在AIM 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 基于单一氨基胺的荧光光谱学研究了其对剪切力的构象反应和 Copepodamide脂质。在AIM 3中，我们将确定RC3相关的生物发光信号途径是否为Gaq- 依赖性，同时使用敲低和敲除实验以及体外测量 GTPase-Glotm分析和[3H]基于GDP的GDP释放分析。 R21项目数据将确定是否 鞭毛藻衍生的GPCR -RC3与剪切力诱导的生物发光有关。项目的 完成将建立一个启动基础，以完全研究GPCR信号的分子机制 在红潮血中，控制或调节开花的出现具有深远的意义。