Prostaglandin I synthase, Thromboxane A synthase & Prostaglandin E synthase

描述（申请人提供）：本项目的总体目标是了解三种类花生酸合成酶，血栓烷A2（TXA 2）合酶（TXAS），前列腺素12（P12）（前列环素，PGI 2）合酶（PGIS），和诱导型微粒体前列腺素E2（PGE 2）合酶-1（mPGES-1）影响它们的酶功能以及它们与上游酶环氧化酶-1（COX-1）和环氧化酶-2（考克斯-2）在TXA 2生物合成中的功能偶联（引起中风和心脏病发作的关键促血栓形成介质）、PGI 2（对抗中风和心脏病发作的关键抗血栓形成介质）和PGE 2（关键促炎介质）。PGIS、mPGES-1和TXAS具有共同的底物前列腺素H2（PGH 2），由考克斯-1或-2产生，主要存在于内质网（ER）膜。目前的研究表明，PGIS和mPGES-1与考克斯-2在功能上偶联，TXAS与COX-2在功能上偶联。与属于P450家族的PGIS和TXAS相比，mPGES-1属于具有不同一级结构和膜拓扑结构的酶家族。这使我们假设PGIS、TXAS和mPGES-1与单个考克斯同种型具有不同的功能偶联模式，并且与ER膜中的PGH 2具有不同的相互作用模式。测定PGH 2从COX向下游酶的移动（呈递）以及它们在ER膜中的物理接近性对于阐明它们不同功能偶联的机制至关重要。根据Pl以前的供资，新的具体目标是：a）。鉴定并比较参与PGH 2呈递的TXAS、PGIS和mPGES-1的膜锚结构域中的结构和关键残基，其不同地影响TXA 2、PGI 2和PGE 2的生物合成; B）。确定mPGES-1的膜拓扑结构和溶液结构以与PGIS和TXAS进行比较;和c）.阐明COX与PGIS、TXAS或mPGES- 1之间的物理接近性，以建立物理分离与其功能耦合之间的关系。这些结果将通过使用综合的生物化学和生物物理方法来实现;例如，重组蛋白和高分辨率NMR光谱。这些研究将提供重要的洞察力，了解控制PGI 2，TXA 2和PGE 2的生物合成的分子机制，介导血管和炎症性疾病，并设计下一代治疗策略。

DESCRIPTION (provided by applicant): The overall goal of this project is to understand how the native, membrane-bound structures of three eicosanoid-synthesizing enzymes, thromboxane A2 (TXA2) synthase (TXAS), prostaglandin 12(prostacyclin, PGI2) synthase (PGIS), and the inducible microsomal prostaglandin E2 (PGE2) synthase-1 (mPGES-1) influence their enzyme functions and their functional coupling with upstream enzymes, cyclooxygenase-1 (COX-l) and -2 (COX-2) in the biosynthesis of TXA2 (a key pro-thrombotic mediator causing stroke and heart attack), PGI2 (a key anti-thrombotic mediator against stroke and heart attack) and PGE2 (a key proinflammatory mediator). PGIS, mPGES-1 and TXAS share a common substrate, prostaglandin H2 (PGH2), produced by COX-1 or -2, mainly occurring in the endoplasmic reticulum (ER) membrane. Current studies have revealed that PGIS and mPGES-1 seem to be functionally coupled with COX-2, and TXAS is functionally coupled with COX-lin the ER membrane. The mPGES-1 belongs to a family of enzymes with a different primary structure and membrane topology compared to that of PGIS and TXAS, belonging to the P450 family. This has led us to hypothesize that PGIS, TXAS and mPGES-1 have distinct modes of functional coupling with individual COX isoforms and distinct modes of interaction with PGH2 in the ER membrane. Determination of PGH2 movement (presentation) from the COXs to the downstream enzymes, and their physical proximities in the ER membrane are crucial to elucidate the mechanisms of their different functional coupling. Based on the Pl's previous funding, the new Specific Aims are proposed to: a). Identify and compare the structures and key residues in the membrane anchor domains of TXAS, PGIS and mPGES-1 involved in the PGH2 presentation influencing their biosynthesis of TXA2, PGI2 and PGE2 differently; b). Determine the membrane topology and solution structure of mPGES-1 for comparison with PGIS and TXAS; and c). Elucidate the physical proximities between the COXs and PGIS, TXAS or mPGES- 1 to establish the relationship of the physical separations and their functional couplings. The results will be achieved by using integrated biochemical and biophysical approaches; such as, recombinant proteins and high resolution NMR spectroscopy. These studies will provide insight important to understanding the molecular mechanisms in controlling the biosynthesis of PGI2, TXA2 and PGE2, which mediates vascular and inflammatory diseases, and designs of next generation therapeutic strategies.