PGIS, TXAS & PGES: STRUCTURE/FUNCTION

描述(申请人提供)：本项目的总体目标是了解血栓素A2(TXA2)合成酶(TXA2)、前列腺素12(PGI2)合成酶(PGIs)和可诱导的微粒体前列腺素E2(PGE2)合成酶-1(mPGES-1)三种二十烷类化合物合成酶的天然膜结合结构是如何影响它们的酶功能以及它们与上游酶环氧合酶-1(COX-L)和环氧合酶-2(COX-2)在TXA2(导致中风和心脏病发作的关键促血栓形成介质)生物合成中的功能耦合。PGI2(中风和心脏病发作的关键抗血栓介质)和PGE2(关键的促炎介质)。PGIs、mPGES-1和Txas共用一个底物前列腺素H2(PGH2)，由COX-1或-2产生，主要存在于内质网(ER)膜上。目前的研究表明，PGIs和mPGES-1似乎与COX-2在功能上偶联，而Txas与COX-LIN在内质网上功能偶联。MPGES-1属于一个与P450家族的PGIs和Txas具有不同一级结构和膜拓扑结构的酶家族。这使得我们假设PGIs、Txas和mPGES-1与不同的COX亚型具有不同的功能偶联模式，并与内质网中的PGH2具有不同的相互作用模式。确定PGH2从Coxs到下游酶的运动(呈现)以及它们在内质网膜上的物理接近对于阐明它们不同的功能偶联机制至关重要。在人民解放运动以前资金的基础上，提出了新的具体目标：a)。鉴定和比较参与PGH2呈递的Txas、PGIs和mPGES-1的膜锚定结构域的结构和关键残基对其合成TXA2、PGI2和PGE2的不同影响；确定mPGES-1的膜拓扑结构和溶液结构，以与PGIs和TxAs进行比较；c)阐明Coxs和PGIs、Txas或mPGES-1之间的物理接近，以建立物理分离和它们的功能偶联之间的关系。这些结果将通过综合使用生化和生物物理方法来实现，例如重组蛋白质和高分辨率核磁共振波谱。这些研究将为理解调节血管和炎症性疾病的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.