PGIS, TXAS & PGES: STRUCTURE /FUNCTION

描述（由申请人提供）：该项目的总体目标是了解三种二十烷类合成酶，血栓素A2 （TXA2）合成酶（TXAS），前列腺素12（prostacyclin, PGI2）合成酶（PGIS）和诱导微粒体前列腺素E2 （PGE2）合成酶-1 （mPGES-1）的天然膜结合结构如何影响它们的酶功能及其与上游酶的功能偶联。环氧化酶-1 （cox -1）和-2 （COX-2）参与TXA2（导致中风和心脏病发作的关键促血栓介质）、PGI2（中风和心脏病发作的关键抗血栓介质）和PGE2（关键促炎症介质）的生物合成。PGIS， mPGES-1和TXAS有一个共同的底物，前列腺素H2 (PGH2)，由COX-1或-2产生，主要发生在内质网（ER）膜上。目前的研究表明，PGIS和mPGES-1似乎与COX-2有功能偶联，TXAS在ER膜上与COX-2有功能偶联。与PGIS和TXAS相比，mPGES-1属于P450家族，具有不同的初级结构和膜拓扑结构。这使得我们假设PGIS、TXAS和mPGES-1与单个COX亚型具有不同的功能偶联模式，并且与内质网膜中的PGH2具有不同的相互作用模式。确定PGH2从cox到下游酶的运动（呈递），以及它们在内质网膜上的物理距离，对于阐明它们不同功能偶联的机制至关重要。在此基础上，我们提出了新的具体目标：a)。鉴定和比较TXAS、PGIS和mPGES-1参与PGH2呈递的膜锚域结构和关键残基对其生物合成TXA2、PGI2和PGE2的影响；b)。确定mPGES-1的膜拓扑结构和溶液结构，并与PGIS和TXAS进行比较；和c)。阐明cox与PGIS、TXAS或mPGES- 1之间的物理接近度，建立物理分离及其功能耦合关系。结果将通过综合使用生化和生物物理方法来实现；如重组蛋白和高分辨率核磁共振波谱。这些研究将为理解介导血管和炎症疾病的PGI2、TXA2和PGE2生物合成的分子机制，以及设计下一代治疗策略提供重要的见解。

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.