PGI & TXA SYNTHASES-MEMBRANE ANCHOR STRUCTURE/FUNCTION

前列腺素I

• 批准号: 6527094
• 负责人: KE-HE RUAN
• 金额: $ 22.36万
• 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 2004-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6527094
• 关键词: active sites; cell membrane; circular dichroism; conformation; cytochrome P450; eicosanoid metabolism; endoplasmic reticulum; enzyme activity; enzyme structure; enzyme substrate; fatty acid biosynthesis; fluorescence microscopy; immunocytochemistry; laboratory rabbit; liposomes; membrane model; membrane structure; microsomes; nuclear magnetic resonance spectroscopy; peptides; prostacyclins; prostaglandin endoperoxide synthase; protein structure function; thromboxanes; vasoconstrictors

DESCRIPTION (Adapted from Abstract) The long-term goal of this project is to understand how the native, membrane-bound structures of two eicosanoid-synthesizing cytochrome P450s, thromboxane A2 synthase (TXAS) and prostaglandin I2 synthase (PGIS), influence enzyme function and coordination with prostaglandin H2 synthase (PGHS), and to understand the membrane topology of mammalian P450 superfamily. TXAS converts prostaglandin H2 (PGH2), produced by PGHS in endoplasmic reticulum. (ER) lumen to thromboxane A2 (TXA2) on the cytoplasmic side of the ER. TXA2 is a mediator with potent platelet aggregatory and vasoconstrictive properties. PGIS converts the same substrate, PGH2, to prostaglandin I2 (PGI2), with biological activities that are opposite to TXA2. TXA2 and PGI2, play important roles in a wide variety of physiological and pathological processes affecting blood and vasculature. Biosynthesis of TXA2 or PGI2 involves coordination of either TXAS or PGIS with PGHS anchored on the opposite side of the ER membrane. This raises the possibility that the membrane anchors influence the coordination. PI's research during past funding indicates that the large cytoplasmic domain of PGIS is anchored to the ER membrane by a single N-terminal anchor segment similar to that in other microsomal P450s, but different from TXAS, which appears to have two membrane anchor segments. The results also indicate that the PGIS N-terminal membrane anchor is near the opening of the substrate access channel and influences enzyme reaction rate. These results led PI to hypothesize that PGIS and TXAS have specific substrate-recognition sites in their N-terminal membrane domains, which facilitate substrate access to their active site channels. PI also suspects that the helix F/G loop of TXAS and PGIS contains a membrane contact region distinct from the N-termini. Crystallographic studies suggest that the catalytic domains of PGHS are anchored to the ER lumen by helices A-D, and thus directly abutting the substrate channel to the ER membrane. To test these hypotheses, Dr. Ruan proposes to analyze and compare the membrane anchor domains of TXAS, PGIS, PGHS and P450 2CI, by a variety of techniques, including immunocytochemistry with domain-specific antibodies, molecular modeling, circular dichroism and NMR. Complete 3D structures of PGIS, TXAS and P450 2C I N-terminal membrane segments will be obtained to provide the solution structures in the membrane environment, complementing existing crystallographic data for P450. The Specific Aims are to: 1) Characterize TXAS and PGIS N-terminal membrane anchor domain which influence the enzyme catalysis, localize the residues important to function and determine the 3D structures of the complex with the interactions between the substrate analog and the membrane domains; 2) Identify membrane contact regions in helix F/G loops of TXAS and PGIS and further define their topology and substrate access channels with respect to the ER membrane; 3) Determine the 3D structure of a synthetic peptide mimicking P450 2C1 N-terminal membrane segment to build a general topology and 3D structural models for microsomal P450s; 4) Determine membrane topology and 3D-solution structure of membrane anchor domains of PGHS-1 and -2 in membrane environment. These studies will provide new insight into how the movement of hydrophobic substrates from membrane compartment to enzyme active sites and between the active sites in case of PGHS/PGIS and PGHS/TXAS is accomplished in an efficient manner within the membrane environment, which complement P450 crystallographic data.

描述（改编自摘要） 这个项目的长期目标是了解当地人， 两种合成类二十烷酸的细胞色素的膜结合结构 P450、血栓素A2合酶（TXAS）和前列腺素I2合酶（PGIS）， 影响酶功能和与前列腺素H2合酶协调 （PGHS），并了解哺乳动物P450超家族的膜拓扑结构。 TXAS转化PGHS在内质网中产生的前列腺素H2（PGH 2）， 网状细胞。(ER)血栓素A2（TXA 2）的细胞质侧的管腔 儿TXA_2是一种具有强血小板聚集和血管收缩作用的介质， 特性. PGIS将相同的底物PGH 2转化为前列腺素I2（PGI 2）， 具有与TXA 2相反的生物活性。TXA 2和PGI 2，播放 在各种生理和病理过程中的重要作用 影响血液和脉管系统 TXA 2或PGI 2的生物合成涉及TXAS或PGIS与 PGHS锚定在ER膜的相对侧。这就提出了一个 膜锚影响协调的可能性。 PI在过去资助期间的研究表明， PGIS通过单个N-末端锚段锚定到ER膜上 与其他微粒体P450类似，但与TXAS不同，TXAS 似乎有两个膜锚段。结果还表明， PGIS N-末端膜锚靠近衬底入口的开口 通道，影响酶反应速率。这些结果导致PI 假设PGIS和TXAS具有特异性底物识别位点， 它们的N-末端膜结构域，其促进底物进入它们的 活跃的网站频道PI还怀疑TXAS和PGIS的螺旋F/G环 含有不同于N-末端的膜接触区。 晶体学研究表明，PGHS的催化结构域是 通过螺旋A-D锚定到ER腔，并因此直接邻接ER腔。 底物通道到ER膜。为了验证这些假设，阮博士 分析比较TXAS、PGIS、PGHS的膜锚结构域 和P450 2CI，通过各种技术，包括免疫细胞化学， 结构域特异性抗体、分子建模、圆二色性和NMR。 PGIS、TXAS和P450 2C I N-末端膜区段的完整3D结构 以提供膜中的溶液结构 环境，补充现有的晶体学数据P450。 具体目的是：1）表征TXAS和PGIS N端膜 锚结构域影响酶的催化作用，定位酶的残基 重要的是功能和确定复杂的三维结构与 底物类似物和膜结构域之间的相互作用; 2）鉴定 TXAS和PGIS的螺旋F/G环中的膜接触区域，并进一步定义 它们相对于ER膜的拓扑结构和底物进入通道; 3)确定模拟P450 2C 1的合成肽的3D结构 N-末端膜片段，以构建一般拓扑结构和3D结构 微粒体P450的模型; 4）确定膜拓扑结构和3D解决方案 膜环境中PGHS-1和-2膜锚结构域结构。 这些研究将为疏水物质的运动提供新的见解 底物从膜隔室到酶活性位点以及 在PGHS/PGIS和PGHS/TXAS的情况下， 一种有效的方式内的膜环境，其中补充P450 晶体学数据