The role of the C1P/cPLA2-alpha interaction in eicosanoid biosynthesis.

描述（由申请人提供）：在本资助申请中，我们专注于脓毒症的生理状况，脓毒症是一个用于描述严重感染引起的严重疾病的术语。脓毒症的死亡率为每年> 215，000名患者，并且脓毒症是美国的第十大死亡原因。每年治疗650，000例新败血症病例的估计年成本为约170亿美元，使得这种疾病成为非常昂贵的医疗状况。已经进行了许多治疗这种疾病的临床试验，但不幸的是，这些试验显示出有限的成功。因此，对治疗该疾病的新疗法存在重大需求。 在这项资助申请中提出的研究与脓毒症直接相关，因为我们正在研究3-PUFA衍生的脂质介质和类花生酸的生物合成途径。这些专门的化学介质的时间和空间产生积极地控制脓毒症导致的过度活跃的炎症反应。类二十烷酸和3-PUFA衍生的脂质介质（二十碳五烯酸（EPA）衍生的脂质介质）的合成始于初始限速步骤，即通过IVA族胞质磷脂酶A2（cPLA 2？）形成花生四烯酸（AA）（类二十烷酸）或EPA衍生的脂质介质形成EPA。1-磷酸神经酰胺（C1 P）是一种具有生物活性的鞘脂，是cPLA 2的直接激活剂。无论是在体外还是在细胞中。此外，从Chalfant实验室发表的研究结果表明，在cPLA 2的C1 P相互作用的关键氨基酸的诱变？抑制酶对几种炎性激动剂的应答易位的能力。因此，C1 P是激活cPLA 2？所必需的，并且是细胞中类花生酸合成的主要调节剂。 为了进一步了解其生理相关性 脂质：蛋白质相互作用在体内，我们的实验室创造了一个敲入小鼠与C1 P相互作用的cPLA 2？消融。我们的初步数据已经证明了cPLA 2？的这种新遗传模型的一些有趣的发现。例如，一些表型报告的cPLA 2？敲除小鼠cPLA 2？敲入小鼠（例如自然流产）（29），而其他表型在cPLA 2？如对LPS和粪便诱导的败血性休克的完全抗性。有趣的是，cPLA 2？敲入小鼠表现出更高水平的抗炎类花生酸，以及增加诱导的抗炎E-resolvins在响应粪便注射相比，cPLA 2？敲除和野生型小鼠。基于这些数据，我们假设，cPLA 2？敲入小鼠是抵抗败血症，由于持续生产抗炎脂质介质所造成的一种新的“脂质类开关”在cPLA 2？底物利用率我们提出的实验将深入探讨这一假设在体外和体内。

DESCRIPTION (provided by applicant): In this grant application, we are focusing on the physiologic condition of sepsis, which is a term used to describe a severe illness arising from serious infection. The mortality rate of sepsis is >215,000 patients a year, and sepsis is the tenth leading cause of death in the US. The estimated yearly cost for treating the 650,000 new cases of sepsis per year is ~17 billion dollars making this disease a very costly medical condition. Numerous clinical trials for the treatment of this disease have been undertaken, but unfortunately, these trials have shown limited success. As such, there is a major need for new therapeutics to treat the disease. The studies proposed in this grant application are directly related to sepsis as we are examining the biosynthetic pathways of 3-PUFA-derived lipid mediators and eicosanoids. The temporal and spatial production of these specialized chemical mediators actively controls the hyperactive inflammatory response as a result of sepsis. The synthesis of eicosanoids and the 3-PUFA-derived lipid mediators, eicosapentaenoic acid (EPA)-derived lipid mediators, begins with the initial rate-limiting step, the formation of arachidonic aid (AA) for eicosanoids or EPA for the EPA derived lipid mediators, via group IVA cytosolic phospholipase A2 (cPLA2?). Ceramide-1-phosphate (C1P) is a bioactive sphingolipid and a direct activator of cPLA2? both in vitro and in cells. Furthermore, published findings from the Chalfant lab demonstrated that mutagenesis of critical amino acids for the C1P interaction in cPLA2? inhibited the ability of enzyme to translocate in response to several inflammatory agonists. Hence, C1P is required for the activation of cPLA2?, and is a major regulator of eicosanoid synthesis in cells. 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 cPLA2? ablated. Our preliminary data has demonstrated some intriguing findings for this new genetic model of cPLA2?. For example, some phenotypes reported for the cPLA2? knockout mouse were not apparent in the cPLA2? knockin mouse (e.g. spontaneous abortion) (29), while other phenotypes were dramatically accentuated in the cPLA2? knockin mouse such as complete resistance to LPS and fecal-induced septic shock. Interestingly, the cPLA2? knockin mouse demonstrated higher levels of anti-inflammatory eicosanoids as well as increased induction of the anti-inflammatory E-resolvins in response to fecal injection as compared to the cPLA2? knockout and wild-type mice. Based on these data, we hypothesize that the cPLA2? knockin mouse is resistant to sepsis due to the sustained production of anti-inflammatory lipid mediators caused by a novel "lipid-class switch" in cPLA2? substrate utilization. Our proposed experiments will explore this hypothesis in depth both ex vivo and in vivo.