PGI & TXA SYNTHASES--MEMBRANE ANCHOR STRUCTURE/FUNCTION

前列腺素I

• 批准号: 2460216
• 负责人: KE-HE RUAN
• 金额: $ 11.04万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-08-01 至 2000-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2460216
• 关键词: active sites; animal tissue; cell membrane; circular dichroism; conformation; cytochrome P450; eicosanoid metabolism; endoplasmic reticulum; enzyme activity; enzyme structure; enzyme substrate; fatty acid biosynthesis; fluorescence microscopy; immunocytochemistry; liposomes; membrane model; membrane structure; microsomes; nuclear magnetic resonance spectroscopy; peptides; prostacyclins; prostaglandin endoperoxide synthase; protein structure function; thromboxanes; vasoconstrictors

DESCRIPTION: (Adapted from Applicant's Abstract) This is a FIRST award application from Dr. Ke-He Ruan. The title of the project is PGI & TXA Synthases: Membrane Anchor Structure/Function. Prostaglandin H2 (PGH2) is converted to Prostaglandin I2 (PGI2) via the catalytic action of Prostaglandin I2 synthase (PGIS). PGI2 is a vasodilator and inhibitor of platelet function. It recently has been employed intravenously for the treatment of pulmonary hypertension. An eicosanoid with actions as a platelet agonist and vasoconstrictor is thromboxane A2 (TXA2). TXA2 also forms from PGH2, via another enzyme, thromboxane A2 synthase (TXAS), a member of the cytochrome P450 superfamily. The structure and function of the N-terminal membrane anchor domains of PGIS and TXAS have not been clearly elucidated. Dr. Ruan has found that TXAS has two separate N-terminal anchor segments and the N- terminus itself faces the cytosol. In contrast, PGIS has 16% sequence identity with TXAS and the hydropathy profile in the N- terminal domain is different. Dr. Ruan suggests that the PGIS membrane anchor structure and the PGIS spatial orientation in relation to the membrane may be different from TXAS. PGH2 is synthesized in the lumen of the endoplasmic reticulum and therefore, the orientation of PGIS and TXAS active sites in relation to the membrane may be important for their conversion of PGH2 into biochemically active eicosanoids with different effects. Other results have shown that the substrate access channel of TXAS faces the ER membrane with the same orientation of the N-terminal membrane anchor region. In this project, attempts will be made to understand how the cytochrome P450 enzymes control eicosanoid biosynthesis. Techniques will include peptidoliposome reconstitution, anti-peptide antibodies which are site- specific, immunocytochemistry, circular dichroism studies, molecular modeling and NMR spectroscopy. The membrane interactions of the N-terminal anchor domains of PGIS and TXAS will be characterized. They will be compared with other cytochrome P450 enzymes in microsomes. The topological arrangement of the catalytic portion of PGIS will be identified in the membrane and it will be compared to that of TXAS. This will also include the determination of the 3D structures of the N-terminal membrane anchor domains of PGIS and TXAS. These will be compared with other microsomal P450s. The residues involved with substrate channel entrance in TXAS and PGIS will be identified. The topological relationship between the substrate channel opening and the ER membrane will be characterized. The experiments should result in the ability to construct a working model for the arrangement of PGIS and comparison with TXAS in the ER membrane. The information should allow for comprehension of similarities and differences between PGIS and TXAS as they relate to the ER membrane and how they coordinate with PGH synthase. Comparisons can then be made between the membrane anchor structure of these two eicosanoid-forming P450s and the anchor structures of other microsomal P450s.

描述：（改编自申请人的摘要）这是第一个奖项 阮克和博士的申请。 项目名称为PGI & TXA 合成酶：膜锚结构/功能。 前列腺素H2 (PGH2) 通过催化作用转化为前列腺素 I2 (PGI2) 前列腺素 I2 合酶 (PGIS)。 PGI2 是一种血管舒张剂和抑制剂 血小板功能。 最近它被用于静脉注射 肺动脉高压的治疗。 具有作用的类二十烷酸 作为血小板激动剂和血管收缩剂的是血栓素 A2 (TXA2)。 TXA2 也由 PGH2 通过另一种酶血栓素 A2 形成 合酶（TXAS），细胞色素 P450 超家族的成员。 这 N端膜锚定结构域的结构和功能 PGIS 和 TXAS 尚未明确阐明。 阮博士发现 TXAS 有两个独立的 N 端锚定段，并且 N- 末端本身面向细胞质。 相比之下，PGIS 为 16% 与 TXAS 的序列同一性以及 N-中的水病特征 终端域不同。 阮博士建议PGIS膜 锚结构和 PGIS 空间方向的关系 膜可能与 TXAS 不同。 PGH2 合成于 内质网的腔，因此，方向 与膜相关的 PGIS 和 TXAS 活性位点可能是 对于将 PGH2 转化为生化活性物质非常重要 具有不同功效的类二十烷酸。 其他结果表明， TXAS 的底物进入通道面向 ER 膜， N 端膜锚定区域的方向相同。 在这个项目中，将尝试了解细胞色素如何 P450 酶控制类二十烷酸生物合成。 技术将包括 肽脂质体重建，抗肽抗体是位点 特异性、免疫细胞化学、圆二色性研究、分子 建模和核磁共振波谱。 膜的相互作用 PGIS 和 TXAS 的 N 端锚定域将得到表征。 它们将与其他细胞色素 P450 酶进行比较 微粒体。 催化部分的拓扑排列 PGIS 将在膜中被识别，并将与 德克萨斯州的。 这还包括 3D 的确定 PGIS 的 N 端膜锚定结构域的结构和 德克萨斯州。 这些将与其他微粒体 P450 进行比较。 这 TXAS 和 PGIS 中与底物通道入口相关的残留物 将被识别。 之间的拓扑关系 将表征底物通道开口和 ER 膜。 实验应该能够构建一个可行的 ER 中 PGIS 布置模型及其与 TXAS 的比较 膜。 该信息应能够帮助理解 PGIS 和 TXAS 之间的异同 ER 膜以及它们如何与 PGH 合酶协调。 比较 然后可以在这两个膜锚结构之间制作 形成类二十烷酸的 P450 和其他微粒体的锚定结构 P450s。