Genetic Regulation of Unconventional Prostaglandin Metabolism

 描述(申请人提供)：前列腺素(PGs)是临床上重要的脂类信号分子，来源于膜磷脂。它们与多种发育、生理和病理生理过程有关，包括受精、 炎症、代谢性疾病、阿尔茨海默氏症和癌症。PG异构体称为异前列腺素，被用作氧化应激的标志物。目前的学说是PG-内源性过氧化物合成酶(即环氧合酶)是唯一能够启动PG合成的酶。然而，这一模型还没有进行过直接测试。COX酶是NSAIDs的靶标，NSAIDs是广泛用于治疗疼痛和炎症的药物。这一应用是由一项意想不到的发现推动的，即线虫和小鼠合成特定的PGF2α立体异构体，而不依赖于COX。线虫PG在受精过程中起着关键作用，并受信息素和营养线索的动态调节。这项建议的目的是描述非COX依赖的PG生物合成机制。中心假设是进化保守的COX非依赖性代谢途径以组织特异性的方式合成前列腺素F1α和前列腺素F2α立体异构体。三个独立的目标被提出来检验中心假说。目的1鉴定环氧合酶非依赖性PG合成所必需的基因。这些研究将使用简单的线虫模型线虫，因为已经开发出一种基因筛选方法来识别这些基因。液-质联用(LC-MS/MS)将测定突变蠕虫中的PG水平。基因替换实验将评估小鼠同源基因的功能保守性。目的2鉴定线虫PG代谢的生化步骤。稳定同位素标记和质谱学方法将鉴定PG代谢物。核磁共振将确定主要蠕虫PG的立体结构。目的3将研究环氧合酶非依赖性PG在小鼠体内的代谢。LC-MS/MS将在COX基因缺失的小鼠幼鼠和分离的组织中鉴定PGs。在COX缺失的胚胎成纤维细胞培养中的药理学研究将测试对非类固醇抗炎药的通路敏感性。一个长期的目标是发展小鼠作为研究COX非依赖性前列腺癌的模型。这些研究将有助于确定PG合成的第二个广泛使用的途径，并为在人类中描绘这一途径提供路线图。这一结果将为发现PG在发育和疾病中的新功能提供遗传学工具。通过改变PG生物化学，将出现开发治疗癌症、青光眼、心血管疾病和其他人类疾病的治疗方法的新途径。

 DESCRIPTION (provided by applicant): Prostaglandins (PGs) are clinically important lipid signaling molecules derived from membrane phospholipids. They have been implicated in a variety of developmental, physiological, and pathophysiological processes, including fertilization, inflammation, metabolic disease, Alzheimer's disease, and cancer. PG isomers called isoprostanes are used as markers of oxidative stress. The current dogma is that PG-endoperoxide synthase (i.e. Cox) enzymes are the sole enzymes capable of initiating PG synthesis. However, this model had not been directly tested. Cox enzymes are targets of NSAIDs, widely used drugs for treating pain and inflammation. This application is driven by the unexpected discovery that C. elegans and mice synthesize specific PGF2α stereoisomers independent of Cox. C. elegans PGs have a critical role in fertilization and are dynamically regulated by pheromones and nutritional cues. The objective of this proposal is to delineate the Cox- independent PG biosynthesis mechanism. The central hypothesis is that an evolutionarily conserved Cox- independent metabolic pathway synthesizes PGF1α and PGF2α stereoisomers in a tissue-specific fashion. Three independent aims are proposed to test the central hypothesis. Aim 1 will identify genes essential for Cox-independent PG synthesis. The simple roundworm model C. elegans will be used for these studies because a genetic screening method has been developed to identify these genes. Liquid chromatography tandem mass spectrometry (LC-MS/MS) will determine PG levels in mutant worms. Gene replacement experiments will assess functional conservation of mouse homologs. Aim 2 will identify biochemical steps in C. elegans PG metabolism. Stable isotope labeling and mass spectrometry methods will identify PG metabolites. Nuclear magnetic resonance will determine stereostructure of major worm PGs. Aim 3 will investigate Cox- independent PG metabolism in mice. LC-MS/MS will identify PGs in Cox null mouse pups and isolated tissues. Pharmacologic studies in Cox null embryonic fibroblast cultures will test pathway sensitivity to NSAIDs. A long- term goal is develop the mouse as a model to study Cox-independent PGs. These studies will help define a second widely used pathway for PG synthesis and provide a roadmap for delineating this pathway in humans. The results will provide genetic tools for discovering new PG functions in development and disease. By transforming PG biochemistry, new avenues will emerge for developing therapeutics to treat cancer, glaucoma, cardiovascular diseases, and other human disorders.