PGI & TXA SYNTHASES-MEMBRANE ANCHOR STRUCTURE/FUNCTION

前列腺素I

• 批准号: 6652426
• 负责人: KE-HE RUAN
• 金额: $ 22.28万
• 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/6652426
• 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原住民， 两种二十烷类合成细胞色素的膜结合结构 P450、血栓素A2合成酶(Txas)和前列腺素I2合成酶(PGIs)； 影响酶功能及其与前列腺素H_2合成酶的配位 (PGHS)，并了解哺乳动物P450超家族的膜拓扑结构。 TXAS在内质中转化PGHS产生的前列腺素H2(PGH2) 网状结构。(ER)管腔到血栓素A2(TXA2)在细胞质一侧 呃。血栓素A2是一种具有强大的血小板聚集和血管收缩作用的介体 属性。PGIs将相同的底物PGH2转化为前列腺素I2(PGI2)， 具有与TXA2相反的生物活性。TXA2和PGI2，播放 在多种生理和病理过程中发挥重要作用 影响血液和血管系统的。 TXA2或PGI2的生物合成涉及TXAs或PGIs与 PGHS定位于内质网膜的对侧。这引发了 膜锚影响配位的可能性。 PI在过去资助期间的研究表明，大的细胞质区域 通过单个N端锚固段将多个PGI锚定到内质网膜上 与其他微粒体P450相似，但不同于Txas，后者 似乎有两个膜锚段。结果还表明， PGIS N端膜锚位于衬底入口的开口附近 通道，并影响酶反应速度。这些结果导致了PI 假设PGIs和Txas在 它们的N-末端膜结构域，这有助于底物进入其 活跃的站点频道。PI还怀疑Txas和Pgis的螺旋F/G环 含有与N-末端不同的膜接触区。 结晶学研究表明，PGHS的催化结构域为 通过螺旋A-D锚定到内质网管腔，从而直接毗邻 底物通道连接到ER膜。为了检验这些假设，阮博士 建议分析和比较TxAs、PGIs、PGHS的膜锚定结构域 和P450 2CI，通过各种技术，包括免疫细胞化学 区域特异性抗体，分子模拟，圆二色谱和核磁共振。 PGIs、Txas和P450 2C I N端膜段的完整三维结构 将获得在膜中提供溶液结构的 环境，补充现有的P450的结晶学数据。 具体目的是：1)表征TxAs和PGIs N-端膜 影响酶催化的锚定结构域，定位残基 对于功能和确定复合体的3D结构非常重要 底物类似物与膜结构域之间的相互作用；2)识别 Txas和Pgis螺旋F/G环中的膜接触区及其进一步确定 它们相对于ER膜的拓扑结构和底物接入通道； 3)确定模拟P450 2c1的合成肽的三维结构 N端膜段构建一般拓扑图和3D结构 微粒体P450的模型；4)确定膜的拓扑结构和3D-解决方案 膜环境中PGHS-1和-2膜锚定结构域的结构 这些研究将为疏水分子的运动提供新的见解 底物从膜室到酶活性部位以及之间 在PGHS/PGIS和PGHS/TxAS情况下的活动站点在 在膜环境中的一种有效方式，补充了P450 结晶数据。