FATTY ACID AND PEPTIDE AMIDATION--A SHARED MECHANSIM

脂肪酸和肽酰胺化——共享机制

• 批准号: 6540029
• 负责人: GREGORY P MUELLER
• 金额: $ 22.28万
• 依托单位: HENRY M. JACKSON FDN FOR THE ADV MIL/MED
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-01 至 2004-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6540029
• 关键词: Adenoviridae; amidation /deamidation; antisense nucleic acid; brain metabolism; cell differentiation; cell free system; cell line; disulfiram; enzyme activity; fatty acid metabolism; isozymes; laboratory rat; neuroblastoma; neurogenesis; neuropharmacology; oxygenases; peptides; protein metabolism; radioimmunoassay; transfection /expression vector

DESCRIPTION: (Adapted from Applicant's Abstract ) Sleep is ubiquitous among mammals and essential for life. More than seventy types of sleep disorders chronically affect millions of Americans in all age groups. Recently, it was shown that the primary fatty-acid amide, oleamide, is a mediator of sleep and may thus contribute to the genesis of sleep disorders. Oleamide accumulates in cerebral spinal fluid during sleep deprivation and induces profound motor quiescence and a sleep-like state upon administration. Primary fatty-acid amides represent a new class of receptor-active signaling molecules whose biogenesis is unknown. Understanding the pathway involved will offer targets for the therapeutic interventions for treating sleep disorders. Recently, we established in vitro that peptidylglycine-alpha-amidating monooxygenase (PAM; EC 1.14.17.3) is able to catalyze the formation of oleamide. PAM is known for its role as the rate-limiting enzyme in the production of peptide messengers and may thus regulate the production of primary fatty-acid amides as well. The objective of this research is to elucidate the mechanisms that govern oleamide production in brain. Three specific aims are proposed for testing the hypothesis that PAM is the physiologic mediator of oleamide biosynthesis. Aim 1. Establish in a cell-free system that oleamide biosynthesis is dependent upon the actions of PAM. Cell-free models permit the direct investigation of fundamental reaction components. Subcellular fractions of N18TG2 mouse neuroblastoma cells, a line which expresses PAM and synthesizes oleamide, will be used to determine the effects of PAM inhibition on the biosynthesis of oleamide. Aim 2. Establish in cell culture that the induction of PAM during neuronal differentiation mediates an increase in oleamide production. Antisense RNA inhibition of PAM expression will be used to directly establish PAM as an integral component of the oleamide biosynthesis pathway in whole cells. Aim 3. Establish that the production of oleamide in vivo is dependent upon the action of PAM. Experiments conducted in rats will take into account the full biologic complexity of the mammalian organism. Direct measures of oleamide in brain and assessments of tissue activity for oleamide biosynthetic labeling will be used to demonstrate the extent to which inhibition of PAM in vivo impairs oleamide biosynthesis. These studies will define the physiologic role of PAM in mediating the biosynthesis of oleamide. Demonstrating that oleamide biosynthesis proceeds through PAM, or via an alternative pathway, will provide a basis for improving the diagnosis and treatment of sleep disorders.

描述：（改编自申请人的摘要）睡眠无处不在 哺乳动物，生命至关重要。超过70种类型的睡眠障碍 长期影响所有年龄段的数百万美国人。最近，是 表明主要的脂肪酸酰胺，Oleamide，是睡眠和 因此，可能有助于睡眠障碍的起源。 Oleamide积累 睡眠剥夺期间的脑脊髓液并诱导深刻的电动机 给药时静止和类似睡眠状态。一级脂肪酸 酰胺代表了一种新的受体活性信号分子 生物发生未知。了解所涉及的路径将提供目标 用于治疗睡眠障碍的治疗干预措施。最近，我们 在体外确定肽基甘氨酸 - α腺苷酶（PAM; EC 1.14.17.3）能够催化Oleamide的形成。帕姆以 它作为限速酶在肽使者生产中的作用 因此，也可能调节原发性脂肪酸酰胺的产生。这 这项研究的目的是阐明支配Oleamide的机制 大脑的产生。提出了三个特定目标以测试 假设PAM是Oleamide生物合成的生理介体。目的 1。在无细胞系统中建立Oleamide生物合成取决于 PAM的行动。无细胞模型允许直接研究 基本反应组成部分。 N18TG2小鼠的亚细胞分数 神经母细胞瘤细胞（表达PAM并合成Oleamide的线条）将会 用于确定PAM抑制对生物合成的影响 Oleamide。目标2。在细胞培养中确定PAM在细胞培养中的诱导 神经元分化介导了Oleamide产生的增加。反义 RNA抑制PAM表达将用于直接建立PAM作为一个 全细胞中Oleamide生物合成途径的整体成分。目标3。 确定在体内的Oleamide的产生取决于作用 帕姆。在大鼠中进行的实验将考虑到完整的生物学 哺乳动物生物的复杂性。直接衡量大脑中的Oleamide和 将使用Oleamide生物合成标记的组织活性评估 为了证明对体内PAM的抑制程度在多大程度上损害了Oleamide 生物合成。这些研究将定义PAM在 介导oleamide的生物合成。证明那个oleamide 生物合成通过PAM或通过替代途径进行 改善睡眠障碍诊断和治疗的基础。