Elucidation of a eukaryotic chemorepulsion mechanism

阐明真核化学排斥机制

基本信息

批准号：
9357616
负责人：
Richard H Gomer
金额：
$ 27.62万
依托单位：
TEXAS A&M UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-09-23 至 2020-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9357616
关键词：
1-Phosphatidylinositol 3-Kinase Actins Adult Respiratory Distress Syndrome Affinity Agonist Bacteria Binding Sites Biochemical Biochemistry Biological Assay Biological Models CT2584 HMS Cell membrane Cell surface Cells Cellular biology Chemotactic Factors Coupled Cyclic AMP Data Decubitus ulcer Development Diabetic ulcer Dictyostelium Dipeptidyl-Peptidase IV Disease Drug Targeting Eukaryotic Cell Face Foundations G-Protein-Coupled Receptors G-substrate GTP-Binding Protein alpha Subunits, Gs Genes Genetic Genetic Screening Heterotrimeric GTP-Binding Proteins Human Inflammation Knock-out Lead Learning Leukocytes Lipids Lung diseases Mediating Modeling Molecular Molecular Biology Morphogenesis Mus Muscle Cells Nomenclature PAR-2 Receptor Pathway interactions Peptide Hydrolases Play Process Proliferating Property Proteinase-Activated Receptors Proteins Recombinants Reporting Rheumatoid Arthritis Shotguns Signal Transduction Signal Transduction Pathway Techniques Testing Therapeutic Tissues Varicose Ulcer Vascular Proliferation Work cell motility cell type extracellular homologous recombination insight mouse model neutrophil polarized cell polymerization protein purification receptor small molecule tumor

项目摘要

There is good evidence that some cells secrete chemorepellents that cause specific cell types to move away from them. However, much remains to be understood about the identity of the chemorepellents, their receptors, and the mechanisms they use to direct cell motility. We found that proliferating Dictyostelium cells secrete a protein called AprA, and that AprA is an extracellular signal that functions as a chemorepellent. Although AprA has little sequence similarity to mammalian proteins, AprA has predicted structural similarity to the human secreted dipeptidyl protease DPPIV, and shares functional properties with DPPIV. We found that human DPPIV is a chemorepellent for human and mouse neutrophils, and when applied locally, DPPIV can induce neutrophils to leave a tissue in two mouse models of a lung disease called acute respiratory distress syndrome (ARDS), and a mouse model of rheumatoid arthritis. To gain insights into a fundamental mechanism used in morphogenesis, ways to induce neutrophils to leave a tissue, and how one could augment or diminish the effect of a chemorepellent, we propose three specific aims to elucidate the molecular mechanisms used by AprA and DPPIV to cause chemorepulsion. Aim 1 is to identify the AprA receptor, since this plays a key role in the Dictyostelium chemorepulsion mechanism. Our preliminary work has identified a predicted G protein- coupled receptor called GrlH as a possible AprA receptor. We will carefully test this, and if GrlH is not the receptor, we will use several approaches to identify the receptor. Aim 2 is to elucidate the AprA chemorepulsion signal transduction pathway. Our preliminary data indicate that some components of the chemorepulsion mechanism are different from components used by the chemoattraction mechanism that allows Dictyostelium cells to aggregate toward cAMP. We will determine the extent to which the chemorepulsion mechanism uses known components of the chemoattraction mechanism, as well as use the power of unbiased genetic screens in Dictyostelium to identify additional components of the chemorepulsion mechanism. Aim 3 is to test the hypothesis that DPPIV uses a G protein-coupled receptor called PAR2 to induce neutrophil chemorepulsion, and use what we learn about the Dictyostelium chemorepulsion mechanism to determine the similarities and differences between the Dictyostelium and the human neutrophil chemorepulsion mechanisms. Together, this work combining molecular biology, genetics, cell biology, and biochemistry will help to elucidate eukaryotic chemorepulsion mechanisms, and identify potential drug targets that could enhance or inhibit chemorepulsion.

有充分的证据表明，一些细胞会分泌化学防护剂，导致特定细胞类型移动远离他们。然而，关于化学驱虫剂的身份、它们的作用还有很多需要了解的地方。受体及其用于指导细胞运动的机制。我们发现增殖的盘基网柄菌细胞分泌一种名为 AprA 的蛋白质，AprA 是一种细胞外信号，具有化学驱避剂的作用。尽管 AprA 与哺乳动物蛋白的序列相似性很少，但 AprA 预测了与哺乳动物蛋白的结构相似性。人类分泌型二肽基蛋白酶 DPPIV，与 DPPIV 具有相同的功能特性。我们发现人 DPPIV 是人和小鼠中性粒细胞的化学驱避剂，当局部应用时，DPPIV 可以诱导中性粒细胞在两种患有急性呼吸窘迫肺部疾病的小鼠模型中留下组织综合征（ARDS）和类风湿关节炎小鼠模型。深入了解基本机制用于形态发生、诱导中性粒细胞离开组织的方法以及如何增强或减弱为了阐明化学驱虫剂的作用，我们提出了三个具体目标来阐明化学驱虫剂使用的分子机制 AprA 和 DPPIV 引起化学排斥。目标 1 是鉴定 AprA 受体，因为它在盘基网柄菌化学脉冲机制。我们的初步工作已经确定了一个预测的 G 蛋白—— 称为 GrlH 的偶联受体可能是 AprA 受体。我们将仔细测试这一点，如果 GrlH 不是受体，我们将使用多种方法来识别受体。目标 2 是阐明 AprA 化学脉冲信号转导途径。我们的初步数据表明，化学脉冲机制与化学吸引机制所使用的成分不同，允许盘基网柄菌细胞向 cAMP 聚集。我们将确定在多大程度上化学脉冲机制使用化学吸引机制的已知组件，以及使用在盘基网柄菌中进行无偏见的遗传筛选来识别化学脉冲的其他成分的能力机制。目标 3 是检验 DPPIV 使用称为 PAR2 的 G 蛋白偶联受体来诱导中性粒细胞化学脉冲，并利用我们对盘基网柄菌化学脉冲机制的了解确定盘基网柄菌和人类中性粒细胞之间的异同化学脉冲机制。这项工作结合了分子生物学、遗传学、细胞生物学和生物化学将有助于阐明真核化学排斥机制，并确定潜在的药物靶点可以增强或抑制化学排斥。