PGIS, TXAS & PGES: STRUCTURE/FUNCTION

德克萨斯州 PGIS

• 批准号: 7111019
• 负责人: KE-HE RUAN
• 金额: $ 25.38万
• 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 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7111019
• 关键词: active sites; cell membrane; chimeric proteins; circular dichroism; conformation; eicosanoid metabolism; endoplasmic reticulum; enzyme activity; enzyme mechanism; enzyme structure; enzyme substrate; fatty acid biosynthesis; fluorescence resonance energy transfer; fluorescence spectrometry; membrane model; membrane structure; nuclear magnetic resonance spectroscopy; oxidoreductase; prostaglandin E; prostaglandin endoperoxide synthase; prostaglandins; protein structure function; site directed mutagenesis; thromboxanes; vasoconstrictors

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.

描述（由申请人提供）：该项目的总体目标是了解三种类菌烷酸合成酶的本机，膜结合结构如何如何如何如何，THOMBOXANE A2（TXA2）合成酶（TXAS）合成酶（TXA）（Prostaglandin 12 Synthase-1（MPGES-1）在TXA2的生物合成中影响其酶功能及其功能耦合，环氧酶-1（Cox-L）和-2（Cox-2）在TXA2的生物合成中（COX-L）和-2（Cox-2）（Cox-l）（COX-2）（一种关键的促进式介体，引起stroke and Mediator，PGI2（PGI），PGI2（pGI2）促炎性调解人）。 PGIS，MPGES-1和TXA具有由Cox-1或-2产生的常见底物，前列腺素H2（PGH2），主要发生在内质网中（ER）膜。当前的研究表明，PGIS和MPGES-1似乎在功能上与COX-2结合，TXAS在功能上与Cox-Lin the ER膜结合。与属于P450家族的PGIS和TXA相比，MPGES-1属于具有不同主要结构和膜拓扑的酶家族。这使我们假设PGI，TXAS和MPGES-1具有与单个COX同工型的功能耦合模式，并且在ER膜中与PGH2的相互作用不同。从Coxs到下游酶的PGH2运动（表现）的测定以及它们在ER膜中的物理接近对于阐明其不同功能耦合的机制至关重要。根据PL先前的资金，提出了新的特定目标：a）。识别并比较PGH2表现中涉及TXA，PGIS和MPGES-1膜锚域中的结构和关键残基，从而不同地影响其TXA2，PGI2和PGE2的生物合成。 b）。确定MPGES-1的膜拓扑和溶液结构，以与PGI和TXA进行比较；和c）。阐明Coxs和PGI，TXAS或MPGES-1之间的物理接近，以建立物理分离及其功能耦合的关系。结果将通过使用综合的生化和生物物理方法来实现。例如重组蛋白和高分辨率NMR光谱。这些研究将为理解控制PGI2，TXA2和PGE2的生物合成的分子机制提供重要的见解，TXA2和PGE2介导了血管和炎症性疾病，以及下一代治疗策略的设计。