The role of the C1P/cPLA2alpha interaction in anaphylaxis

描述(申请人提供)：在这项拨款申请中，我们关注的是过敏反应的生理条件，这是一种严重的全身过敏反应，一旦接触到过敏原就会迅速发生。发病率已大幅上升至每10万人中有49.8人，其中许多是患有心血管疾病等并存疾病的老年人。这项拨款申请中建议的研究与过敏反应直接相关，因为我们正在研究二十烷类化合物和其他脂质介体的生物合成途径。众所周知，这些生物活性脂类参与了炎症途径，以及在过敏反应中介导肥大细胞的激活和脱颗粒。二十烷类化合物的合成始于最初的限速步骤，即通过IVA胞浆磷脂酶A2(CPLA2a)形成花生四烯酸(AA)。神经酰胺-1-磷酸(C1P)是一种生物活性的鞘磷脂，在体外和细胞内都是cPLA2a的直接激活剂。C1P相互作用的关键氨基酸的突变抑制了酶对几种炎性激动剂的反应能力。因此，cPLA2a与C1P的结合是生物活性脂质介体(如二十烷类化合物)生物合成中的一个重要调节事件。为了进一步了解这种脂质的生理相关性：体内的蛋白质相互作用， 我们实验室创造了一种敲打小鼠，其C1P相互作用位点cPLA2a被去除。我们的初步数据已经为cPLA2a的这种新的遗传模型展示了一些有趣的发现。例如，报告的cPLA2a基因敲除小鼠的一些表型在cPLA2a敲除小鼠中不明显(例如自然流产)[14]，而其他表型被授予cPLA2a敲除小鼠，如对被动全身过敏反应的抵抗力。基于我们实验室的这些初步发现和文献的推断，我们假设cPLA2a敲击小鼠由于失去特定的脂质介质而对过敏反应产生抵抗力。我们进一步假设，尽管野生型小鼠的过敏表型随着年龄的增长而增加，但cPLA2a敲击小鼠仍将保持抵抗力。我们提议的实验将在体外和体内深入探索这一假说，希望开发新的疗法来对抗年轻人和老年人口的这种疾病状态。

DESCRIPTION (provided by applicant): In this grant application, we are focusing on the physiological condition of anaphylaxis, which is a severe, systemic allergic reaction that occurs quickly upon exposure to an allergen. The incidence rate has increased dramatically to 49.8 people per 100,000, many of which are elderly with comorbid conditions such as cardiovascular disease. The studies proposed in this grant application are directly related to anaphylaxis as we are examining the biosynthetic pathways of eicosanoids and other lipid mediators. These bioactive lipids are well known to be involved in inflammatory pathways as well as mediating mast cell activation and degranulation during anaphylactic responses. The synthesis of eicosanoids begins with the initial rate-limiting step, the formation of arachidonic acid (AA) via group IVA cytosolic phospholipase A2 (cPLA2a). Ceramide-1- phosphate (C1P) is a bioactive sphingolipid and a direct activator of cPLA2a both in vitro and in cells. Mutagenesis of critical amino acids for C1P interaction in cPLA2a inhibited the ability of enzyme to translocate in response to several inflammatory agonists. Hence, the association of cPLA2a with C1P is a major regulatory event in the biosynthesis of bioactive lipid mediators (e.g. eicosanoids). To further our understanding of the physiological relevance of this lipid:protein interaction in vivo, our laboratory created a knockin mouse with the C1P interaction site of cPLA2a ablated. Our preliminary data has demonstrated some intriguing findings for this new genetic model of cPLA2a. For example, some phenotypes reported for the cPLA2a knockout mouse were not apparent in the cPLA2a knockin mouse (e.g. spontaneous abortion) [14], while other phenotypes were conferred to the cPLA2a knockin mouse such as resistance to passive systemic anaphylaxis. Based on these preliminary findings by our laboratory and inferences from the literature, we hypothesize that the cPLA2a knockin mouse is resistant to anaphylaxis due to loss of specific lipid mediators. We further hypothesize that though wild type mice have an increased anaphylactic phenotype as they age, the cPLA2a knockin mice will remain resistant. Our proposed experiments will explore this hypothesis in depth both ex vivo and in vivo with the hope of developing new therapeutics to combat this disease state in both young and aging populations.