Elucidation of a eukaryotic chemorepulsion mechanism

阐明真核化学排斥机制

• 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
• 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

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.

有很好的证据表明，一些细胞分泌的化学物质会导致特定类型的细胞移动 远离他们。然而，关于化学抵抗剂的身份，他们的 受体，以及它们用来指导细胞运动的机制。我们发现Dictyostelius增殖的细胞 分泌一种叫做APRA的蛋白质，APRA是一种细胞外信号，起着化学排斥剂的作用。 尽管APRA与哺乳动物蛋白质的序列相似性很小，但APRA预测了结构上的相似性 人分泌的二肽基蛋白酶DPPIV，与DPPIV具有相同的功能特性。我们发现 人DPPIV是人和鼠中性粒细胞的化学抵抗剂，当局部应用时，DPPIV可以 在两种称为急性呼吸窘迫的肺部疾病小鼠模型中，诱导中性粒细胞离开组织 综合征(ARDS)和类风湿性关节炎的小鼠模型。深入了解一种基本机制 用于形态发生，诱导中性粒细胞离开组织的方法，以及如何增加或减少 对于化学排泄剂的作用，我们提出了三个具体的目标来阐明 APRA和DPPIV引起化学排斥。目标1是识别APRA受体，因为这在 网柄苔藓的化学排斥机制。我们的初步工作已经确定了一种预测的G蛋白- 偶联受体Gr1H可能是一种APRA受体。我们将仔细测试这一点，如果GrlH不是 对于受体，我们将使用几种方法来识别受体。目的2是为了阐明APRA 化学排斥信号转导通路。我们的初步数据表明， 化学排斥机制不同于化学吸引机制所使用的组件 使网生植物细胞向cAMP聚集。我们将确定在多大程度上 化学排斥机制使用化学吸引机制的已知组件，以及使用 网柄苔藓中无偏遗传筛选识别化学排斥的额外成分的能力 机制。目标3是检验DPPIV使用名为PAR2的G蛋白偶联受体来 诱导中性粒细胞化学排斥，并利用我们所了解的Dictyostants化学排斥机制 确定网盘基菌和人中性粒细胞的异同 化学排斥机制。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细胞的化学排斥机制，并确定潜在的药物靶点。 这可能会增强或抑制化学斥力。