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

描述(由申请人提供)：血液白细胞在缺血发作后向炎症部位迁移(有益的影响)或向受损的组织迁移(有害的影响)，例如在心脏缺血/再灌注损伤(I/RI)的情况下。在I/R中，当血液供应恢复到受影响的区域时，中性粒细胞通过大量释放活性氧物种(ROS)对健康组织造成损害。我们发现磷脂酶D2(PLD2)对中性粒细胞具有趋化作用。除此之外，我们提出了三条主要的证据，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中存在着意想不到的全球环境基金活性。由于靶基因rac2 Rho GTPase负责细胞的运动，因此PLD2可能是一种主要的全球环境基金介导的趋化作用。由于GEF不是结构性活性的，而是受到严格监管的，我们在AIM 1中建议研究PLD2新的全球环境基金活性的调节。该假说认为PLD2蛋白的活性受酪氨酸磷酸化和与PDZ结构域蛋白相互作用的调节，从而导致PLD2的Px和PH结构域与磷脂酸(PA)共同作用，以确保GTP/GDP交换活性。候选蛋白为VEGFR2和JAK3，而PDZ结构域蛋白为MUPP1和Mda9/Syntenin。(2)第二，我们提出了PLD2在细胞信号转导过程中作为蛋白质网络中心的初步证据。我们建议在AIM 2中研究PLD2和14-3-3相互作用的新途径，以防止GDF-15在缺血再灌注损伤条件下的趋化作用。我们将通过生化、遗传学(rac2-/-、Wasp-/-和PLD2-/-KO小鼠)和荧光显微镜实验来研究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体的形成，我们将量化这些信号分子在趋化和类似I/RI条件下的空间组织。(3)在小鼠心肌/再灌注损伤模型中，PLD抑制剂保护心脏免受缺血/再灌流损伤，使PLD处于该疾病的中心。在AIM 3中，我们假设从白细胞渗入缺血/梗死区的PLD将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小鼠以及用特定PLD抑制剂植入渗透泵的小鼠身上测试这一假设。我们还将确定PLD在体内运行的信号转导的调节机制。我们期望证明，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。这笔赠款将发现可以在药物上利用的靶点，以减少炎症介导的心脏损伤中中性粒细胞的有害存在。如果我们能避免这些细胞在缺血后过早出现，心力衰竭 然后，心肌梗死就会减少。

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.