PGI & TXA SYNTHASES--MEMBRANE ANCHOR STRUCTURE/FUNCTION

描述：(改编自申请者摘要)这是第一个奖项 阮克和博士的申请书。该项目的名称为PGI&TXA 合成酶：膜锚定结构/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合成酶(PGIs)。前列腺素I2是一种血管扩张剂和抑制剂 血小板功能的变化。它最近被静脉注射用于 肺动脉高压的治疗。有动作的二十烷类化合物 血栓素A2(TXA2)是一种血小板激动剂和血管收缩药。 TXA2也是由PGH2通过另一种酶--血栓素A2形成的 合酶(Txas)，细胞色素P450超家族成员。这个 N-末端膜锚定结构域的结构与功能 PGIs和Txas还没有被清楚地阐明。阮博士发现了 TxAS具有两个独立的N端锚固段，并且N- 终末本身面对胞浆。相比之下，PGI的比例为16% 与Txas的序列同源性和N- 终端域不同。阮博士认为PGIS膜 锚固结构和PGIS的空间定向与 膜可能与Txas不同。PGH2的合成是在 内质网的管腔，因此，内质网的定位 与膜有关的PGIs和Txas活性部位可能是 对它们将PGH2转化为生物化学活性很重要 具有不同作用的二十烷类化合物。其他结果表明， TxAS的衬底接入通道面向电流变膜， N-端膜锚定区的取向相同。 在这个项目中，我们将尝试理解细胞色素是如何 P450酶控制二十烷类化合物的生物合成。技术将包括 肽多脂体重组，抗肽抗体，这些抗体是- 特异性，免疫细胞化学，圆二色谱研究，分子 建模和核磁共振波谱。膜的相互作用 将表征PGIs和TxAs的N-末端锚定域。 它们将与其他细胞色素P450酶在 微粒体。的催化部分的拓扑排列 将在膜中鉴定PGIs，并将其与 Txas的。这也将包括3D的确定 Pgis和Pgis的N-端膜锚定域的结构 德克萨斯。这些将与其他微粒体P450进行比较。这个 Txas和PGIs中与底物通道进入有关的残留物 将会被确认。之间的拓扑关系 底物通道的开通和电流变膜的表征。 实验的结果应该是构建一个工作的 ER中PGI的布局模型及其与TxAS的比较 薄膜。该信息应允许理解 PGIs和Txas的异同在于它们与 ER膜及其与PGH合成酶的相互作用。比较 然后可以制作这两者之间的薄膜锚固结构 二十烷类化合物P450与其他微生物体的锚定结构 P450。

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.