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

C1P/cPLA2-α 相互作用在类二十烷酸生物合成中的作用。

• 批准号: 8716449
• 负责人: Lauren Alexis Hoeferlin
• 金额: $ 5.33万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-03-25 至 2016-03-24
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8716449
• 关键词: Agonist; Amino Acid Sequence; Amino Acids; Anabolism; Anti-Inflammatory Agents; Anti-inflammatory; Applications Grants; Arachidonic Acids; Binding; C2 Domain; Calcium; Cause of Death; Cells; Chemicals; Clinical Treatment; Clinical Trials; Cytosolic Phospholipase A2; Data; Disease; Docosahexaenoic Acids; Eicosanoids; Eicosapentaenoic Acid; Enzymes; Genetic Models; Immunoglobulin Class Switching; In Vitro; Infection; Inflammatory; Inflammatory Response; Injection of therapeutic agent; Ionophores; Knock-out; Knockout Mice; Laboratories; Leukotrienes; Lipids; Mediator of activation protein; Medical; Modeling; Mus; Mutagenesis; Pathogenesis; Pathway interactions; Patients; Phenotype; Phospholipase; Phospholipase A2; Phospholipids; Physiological; Production; Prostaglandins; Prostaglandins I; Proteins; Publishing; Reporting; Resistance; Role; Sepsis; Septic Shock; Septicemia; Site; Sphingolipids; Spontaneous abortion; Thromboxanes; Wild Type Mouse; base; ceramide 1-phosphate; cost; human PLA2G4A protein; in vivo; lipid mediator; mortality; novel; novel therapeutics; pre-clinical; public health relevance; research study; response; success

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.

描述（由申请人提供）：在本拨款申请中，我们重点关注败血症的生理状况，败血症是一个用于描述由严重感染引起的严重疾病的术语。败血症的死亡率每年超过 215,000 名患者，败血症是美国第十大死因。每年治疗 650,000 例脓毒症新病例的估计费用约为 170 亿美元，使这种疾病成为一种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，这些试验的成功有限。因此，非常需要新的疗法来治疗该疾病。 本拨款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二十烷酸的生物合成途径。这些特殊化学介质的时间和空间产生积极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从初始限速步骤开始，即为类二十烷酸形成花生四烯酸 (AA) 或为 EPA 衍生的脂质形成 EPA介质，通过 IVA 组胞质磷脂酶 A2 (cPLA2?)。 Ceramide-1-磷酸 (C1P) 是一种生物活性鞘脂，也是 cPLA2? 的直接激活剂。在体外和细胞内。此外，Chalfant 实验室发表的研究结果表明，cPLA2 中 C1P 相互作用的关键氨基酸发生突变？抑制酶响应几种炎症激动剂而移位的能力。因此，C1P 是 cPLA2 激活所必需的，并且是细胞中类二十烷酸合成的主要调节因子。 为了进一步了解这一现象的生理相关性 体内脂质：蛋白质相互作用，我们实验室创建了具有 cPLA2? C1P 相互作用位点的敲入小鼠？消融。我们的初步数据证明了这种新的 cPLA2 遗传模型的一些有趣的发现。例如，报告的 cPLA2 的一些表型？敲除小鼠在 cPLA2 中不明显？敲入小鼠（例如自然流产）（29），而其他表型在 cPLA2 中显着增强？敲入小鼠，例如对脂多糖和粪便引起的感染性休克的完全抵抗。有趣的是，cPLA2？与 cPLA2? 相比，敲入小鼠表现出更高水平的抗炎类二十烷酸，并且响应粪便注射而增加了抗炎 E-resolvins 的诱导。基因敲除小鼠和野生型小鼠。根据这些数据，我们假设 cPLA2？敲入小鼠对脓毒症具有抵抗力，因为 cPLA2 中新型“脂质类开关”导致抗炎脂质介质持续产生？基材利用率。我们提出的实验将在体外和体内深入探索这一假设。