PLD2, as a GEF or as a Lipase, is Central to Leukocyte Chemotaxis

PLD2 作为 GEF 或脂肪酶，是白细胞趋化性的核心

• 批准号: 8441684
• 负责人: JULIAN G. CAMBRONERO
• 金额: $ 37.46万
• 依托单位: WRIGHT STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-04-01 至 2017-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8441684
• 关键词: Actins; Adaptor Signaling Protein; Affect; Animal Model; Area; Binding; Biochemical; Biochemical Genetics; Blood flow; Cause of Death; Cells; Cellular Assay; Chemotactic Factors; Chemotaxis; D Cells; Data; Disease; Ensure; Enzymes; Fluorescence Microscopy; Fluorescence Resonance Energy Transfer; GDF15 gene; Goals; Grant; Guanosine Triphosphate; Heart; Heart Injuries; Heart failure; Human Pathology; Imagery; Implant; In Vitro; Infarction; Inflammation; Injury; Ischemia; JAK3 gene; Laboratories; Lead; Leukocyte Chemotaxis; Leukocytes; Life; Lipase; Mediating; Modeling; Molecular; Movement; Mus; Myocardial; Myocardial Infarction; Myocardial Ischemia; Myocardial Reperfusion Injury; Neutrophil Infiltration; Nutrient; Oxygen; PH Domain; Pathway interactions; Patients; Phosphatidic Acid; Phospholipase D; Phosphotransferases; Proteins; Pump; Reactive Oxygen Species; Regulation; Reperfusion Injury; Reperfusion Therapy; Research; Resolution; Role; Signal Pathway; Signal Transduction; Signaling Molecule; Signaling Protein; Site; Tertiary Protein Structure; Testing; Therapeutic; Thrombolytic Therapy; Time; Tissues; Tyrosine Phosphorylation; United States; Vascular Endothelial Growth Factor Receptor-2; Vascular blood supply; base; cell motility; cellular imaging; cohort; cytokine; deprivation; human FRAP1 protein; in vivo; inhibitor/antagonist; neutrophil; novel; phospholipase D2; prevent; protein complex; public health relevance; research study; rho GTP-Binding Proteins

DESCRIPTION (provided by applicant): Blood leukocytes migrate towards the sites of inflammation (a beneficial effect) or towards compromised tissues after an ischemic attack (a deleterious effect), such as in the case of ischemia/reperfusion injury (I/RI) in the heart. In I/R, when the supply of blood is restored to the affected area, neutrophils cause damage to healthy tissue via the massive release of reactive oxygen species (ROS). We have discovered that the enzyme Phospholipase D2 (PLD2) is a chemoattractant for neutrophils. In addition to this, we present three major lines of evidence that have allowed us to propose three Specific Aims. (1) First, novel data in our lab has revealed an unexpected GEF activity that exists in the lipase PLD2. Since the target, Rac2 Rho GTPase is responsible for cell movement PLD2 might be a major GEF mediating chemotaxis. Because GEFs are not constitutively active but are kept under tight regulation, we propose in AIM 1 to investigate the regulation of PLD2 novel GEF activity. The hypothesis is that PLD2 GEF activity is regulated by tyrosine phosphorylation and by interaction with PDZ domain proteins, which lead to PX and PH domains of PLD2 and phosphatidic acid (PA) combined action to ensure a GTP/GDP exchange activity. The candidate kinases are VEGFR2 and JAK3, while the PDZ-Domain proteins are MUPP1 and Mda9/Syntenin. (2) Second, we present preliminary evidence of PLD2 as the center of a protein network during cell signaling. We propose in AIM 2 to study a new pathway through which PLD2 and 14-3-3 interact to prevent chemotaxis in ischemia reperfusion injury conditions via GDF-15. We will study the molecular interactions involving PLD2, Rac2, WASp, and the adaptors Grb2 and 14-3-3 through biochemical, genetic (Rac2-/-, WASp-/- and PLD2-/- KO mice) and fluorescence microscopy experiments. Through visualization of protein complex formation in real time by FRET, we will quantify the spatial organization of these signaling molecules during chemotaxis and in I/RI-like conditions. (3) Third, PLD inhibitors protect the heart from injury caused by ischemia/reperfusion in a Murine myocardial/reperfusion injury model, placing PLD at the center of this disease. In AIM 3 we hypothesize that PLD from leukocytes that infiltrate an ischemia/infarct area will have a deleterious effect on the heart conducive to the exacerbation of the injury in vivo. We will test this hypothesis in wild type and PLD2- /- KO mice, as well as in osmotic pump-implanted mice with specific PLD inhibitors. We will also ascertain the PLD regulatory mechanisms of signal transduction that operate in vivo. We expect to demonstrate that depriving PLD confers myocardial protection, with therapeutic potential in conjunction with currently used thrombolytic therapy. This grant will uncover targets that can be exploited pharmacologically to diminish the harmful presence of neutrophils in inflammation-mediated heart injury. If we can avoid the untimely presence of these cells in ischemia, heart failure after myocardial infarction would then be diminished.

描述（由申请人提供）：血液白细胞向炎症部位（有益作用）或缺血发作后受损组织（有害作用）迁移，例如在心脏缺血/再灌注损伤（I/RI）的情况下。在I/R中，当受影响区域的血液供应恢复时，中性粒细胞通过大量释放活性氧（ROS）对健康组织造成损害。我们已经发现磷脂酶D2 （PLD2）是中性粒细胞的化学引诱剂。除此之外，我们提出了三条主要的证据线，使我们能够提出三个具体目标。(1)首先，我们实验室的新数据揭示了脂肪酶PLD2中存在意想不到的GEF活性。由于目标，Rac2 Rho GTPase负责细胞运动，PLD2可能是GEF介导的主要趋化性。由于GEF不具有组成活性，但受到严格的调控，我们在AIM 1中提出研究PLD2新GEF活性的调控。假设PLD2 GEF活性受酪氨酸磷酸化和与PDZ结构域蛋白的相互作用调节，PDZ结构域蛋白导致PLD2的PX和PH结构域与磷脂酸（PA）联合作用，以确保GTP/GDP交换活性。候选激酶是VEGFR2和JAK3，而PDZ-Domain蛋白是MUPP1和Mda9/Syntenin。(2)其次，我们提出了PLD2在细胞信号传导过程中作为蛋白质网络中心的初步证据。我们在AIM 2中提出研究PLD2和14-3-3通过GDF-15相互作用阻止缺血再灌注损伤条件下趋化的新途径。我们将通过生化、遗传学（Rac2-/-、WASp-/-和PLD2-/- KO小鼠）和荧光显微镜实验，研究PLD2、Rac2、WASp和接头Grb2和14-3-3的分子相互作用。通过FRET实时可视化蛋白质复合物的形成，我们将量化这些信号分子在趋化性和I/ ri样条件下的空间组织。(3)第三，在小鼠心肌/再灌注损伤模型中，PLD抑制剂可保护心脏免受缺血/再灌注损伤，将PLD置于该疾病的中心。在AIM 3中，我们假设浸润缺血/梗死区域的白细胞PLD会对心脏产生有害影响，有利于体内损伤的加剧。我们将在野生型和PLD2- /- KO小鼠以及植入特定PLD抑制剂的渗透泵小鼠中验证这一假设。我们还将确定PLD在体内运作的信号转导的调节机制。我们希望证明剥夺PLD可以提供心肌保护，与目前使用的溶栓治疗相结合具有治疗潜力。这项资助将发现靶标，可以利用药理学来减少中性粒细胞在炎症介导的心脏损伤中的有害存在。如果能避免这些细胞过早出现在缺血后，心力衰竭