LIPID PHOSPHATASES AND CANNABINOID BIOSYNTHESIS

脂质磷酸酶和大麻素生物合成

• 批准号: 2861933
• 负责人: Shelley B Hooks
• 金额: $ 1.99万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-03-01 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/2861933
• 关键词: active sites; brain; brain metabolism; cannabinoid receptor; cell line; enzyme activity; enzyme mechanism; enzyme substrate; high performance liquid chromatography; in situ hybridization; isozymes; laboratory rat; lipid biosynthesis; liquid chromatography; mass spectrometry; messenger RNA; phosphatidate phosphatase; phospholipase C; protein localization; radiotracer; thin layer chromatography

Cannabinoid receptors mediate the psychotropic effects of marijuana, as well as those of therapeutic potential, including antinociceptive and immunomodulatory responses. Acylethanolamide (AEA) and 2-monoacyl glycerol (2-MAG) are endogenous cannabinomimetics, and are simply the nonphosphorylated counterparts of the LPA receptor agonists N-acyl ethyl phosphoric acid (NAEPA) and sn-2 LPA, respectively. Further, the structural similarity between the two classes of agonists suggests that sn-2 LPA and NAEPA may be cannabinoid precursors. The characterization of membrane bound phosphatidic acid phosphatases (PAP-2) suggests that these enzymes may provide the enzymatic link between the two classes of lipids. This proposal is designed to test the hypothesis that the biosynthesis of the cannabinomimetics 2-MAG and AEA occurs via PAP-2 mediated hydrolysis of LPA and NAEPA, and the latter is in turn the product of phospholipase C cleavage of N-acyl phosphatidylethanolamine (NAPE). First, the pharmacological and biochemical characterization of PAP-2 isoforms will be extended by determining if sn-2 LPA and NAEPA with long chain, polyunsaturated fatty acyl moities are PAP-2 substrates. Additionally, the predicted extracellular orientation of the PAP-2 active site will be tested experimentally, and structural analogs of PAP-2 substrates will be screened as blockers of PAP-2 activity. Secondly, mass spectroscopy will be employed to determine if NAEPA is detectable in rat brain and if it is present at sufficiently high quantities to support the proposed role as LPA receptor agonist and AEA precursor. Next, neuronal cell lines homogenates will be assayed for the presence of a PLC activity using radiolabelled NAPE as substrate. Finally, to examine whether the generation of cannabinoid ligands by PAP-2s may be physiologically relevant, in situ hybridization will be used to determine if the phosphatases are expressed in the same brain regions as cannabinoid receptors. Herein, a novel biosynthetic route for endogenous cannabinoid ligands is proposed, which may be functionally coupled to a second family of bioactive lipids, the LPA receptor agonists.

大麻素受体介导大麻的精神作用， 以及具有治疗潜力的那些，包括抗伤害感受的， 免疫调节反应。 酰基乙醇酰胺（AEA）和2-单酰基 甘油（2-MAG）是内源性拟大麻素， LPA受体激动剂N-酰基乙基的非磷酸化对应物 磷酸（NAEPA）和sn-2 LPA。此夕h 两类激动剂之间的结构相似性表明， sn-2 LPA和NAEPA可能是大麻素前体。 表征 膜结合磷脂酸磷酸酶（PAP-2）的研究表明， 这些酶可以提供两类酶之间的酶促连接 脂质。 这项提议旨在检验以下假设： 拟大麻素2-MAG和AEA的生物合成通过PAP-2发生 介导的LPA和NAEPA的水解，后者又是 磷脂酶C裂解N-酰基磷脂酰乙醇胺的产物 （NAPE）。 首先，药理学和生物化学表征 PAP-2亚型将通过确定sn-2 LPA和NAEPA 具有长链的多不饱和脂肪酰基部分为PAP-2 印刷受体.此外，预测的细胞外取向的 PAP-2活性位点将进行实验测试，结构类似物 的PAP-2底物将被筛选为PAP-2活性的阻断剂。 其次，将采用质谱法来确定NAEPA是否是 在大鼠大脑中可检测到，并且如果其含量足够高 数量，以支持LPA受体激动剂和AEA的拟定作用 先驱 接下来，将测定神经元细胞系匀浆中的 使用放射性标记的NAPE作为底物，存在PLC活性。 最后，为了检查大麻素配体的产生是否通过 PAP-2可能与生理学相关，原位杂交将被应用于临床。 用于确定磷酸酶是否在同一大脑中表达 作为大麻素受体。在此，提供了用于制备抗肿瘤药物的新的生物合成途径。 内源性大麻素配体的建议，这可能是功能 与第二个生物活性脂质家族LPA受体偶联 激动剂