Genetic Regulation of Unconventional Prostaglandin Metabolism

 描述（由申请人提供）：前列腺素（PG）是来自膜磷脂的临床重要脂质信号分子。它们与多种发育、生理和病理生理过程有关，包括受精、 炎症、代谢疾病、阿尔茨海默病和癌症。称为异前列烷的PG异构体被用作氧化应激的标志物。目前的教条是PG-内过氧化物合酶（即考克斯）酶是能够引发PG合成的唯一酶。然而，这一模式尚未得到直接测试。考克斯酶是NSAID的靶点，NSAID是广泛用于治疗疼痛和炎症的药物。这个应用程序是由一个意外的发现驱动的，即C。线虫和小鼠合成特异性PGF 2 α立体异构体，不依赖于考克斯。C.线虫PGs在受精中具有关键作用，并受信息素和营养线索的动态调节。该建议的目的是描述不依赖于考克斯的PG生物合成机制。中心假设是进化上保守的考克斯非依赖性代谢途径以组织特异性方式合成PGF 1 α和PGF 2 α立体异构体。提出了三个独立的目标来检验中心假设。目的1将确定必需的基因Cox非依赖性PG的合成。简单蛔虫C型。因为已经开发了一种遗传筛选方法来鉴定这些基因，所以将使用秀丽隐杆线虫进行这些研究。液相色谱串联质谱法（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物的功能保守性。目的2将确定C. PG代谢。稳定同位素标记和质谱法将鉴定PG代谢物。核磁共振将确定主要蠕虫PG的立体结构。目的3研究PG在小鼠体内的非考克斯依赖性代谢。LC-MS/MS将鉴别考克斯基因敲除小鼠幼仔和分离组织中的PG。考克斯无效胚胎成纤维细胞培养物的药理学研究将测试对NSAID的途径敏感性。长期目标是开发小鼠作为研究Cox非依赖性PG的模型。这些研究将有助于定义第二个广泛使用的PG合成途径，并为描绘人类的这一途径提供路线图。这些结果将为发现PG在发育和疾病中的新功能提供遗传工具。通过改变PG生物化学，将出现新的途径来开发治疗癌症，青光眼，心血管疾病和其他人类疾病的疗法。

 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.