Catalysis by Prostaglandin Endoperoxide H Synthases

前列腺素内过氧化物 H 合成酶的催化

• 批准号: 7491053
• 负责人: William L Smith
• 金额: $ 44.02万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-01 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7491053
• 关键词: Abbreviations; Acetates; Amino Acids; Anabolism; Antibiotic A23187; Arachidonic Acids; Be++ element; Beryllium; Biochemical; Biology; Bolus Infusion; Bradykinin; Buthionine Sulfoximine; Catalysis; Cell Cycle; Cell Differentiation process; Cells; Commit; Coxibs; Cultured Cells; Cysteine; Cytosolic Phospholipase A2; Diet; Dinoprostone; Docosahexaenoic Acids; EMSA; Eicosapentaenoic Acid; Eicosatetraenoic Acids; Electrophoretic Mobility Shift Assay; Elements; Embryo; Endoglycosidases; Engineering; Environment; Esters; Event; Fatty Acids; Fibroblasts; Fish Oils; Flurbiprofen; Genes; Glycerophospholipids; Goals; HTATIP gene; Health Benefit; Housekeeping; Human; Hydrogen Peroxide; In Vitro; Ionophores; Kidney; Kinetics; Knock-in Mouse; Knockout Mice; Learning; Linoleic Acids; Mammalian Cell; Measures; Mus; NIH 3T3 Cells; Non-Steroidal Anti-Inflammatory Agents; Omega-6 Fatty Acids; Outcome Study; PGF gene; PLA2G4A gene; PPIX; PTGS1 gene; PTGS2 gene; Pathway interactions; Penicillins; Peroxidase; Peroxidases; Personal Satisfaction; Phase; Phenotype; Phospholipids; Placental Growth Factor; Platelet-Derived Growth Factor; Property; Prostaglandin Endoperoxides; Prostaglandin-Endoperoxide Synthase; Prostaglandins; Prostaglandins D; Prostaglandins E; Prostaglandins F; Protein Isoforms; Protein Overexpression; Protoporphyrins; Reduced Glutathione; Regulation; Research; Research Personnel; Role; Series; Stimulus; Streptomycin; System; Testing; Tetradecanoylphorbol Acetate; Thromboxane Receptor; Thromboxanes; Time; concept; cyclooxygenase 1; cyclooxygenase 2; design; feeding; human PLA2G4A protein; human prostaglandin D2 receptor; immunocytochemistry; in vivo; kifunensine; mead acid; mouse PGE synthase 1; phorbol-12-myristate; progesterone 11-hemisuccinate-(2-iodohistamine); programs; prostaglandin R2 D-isomerase; protein degradation; research study; urinary

DESCRIPTION (provided by applicant): The long-term goal of our studies is to understand how prostaglandin (PG) synthesis is regulated. There are two PGH synthases (PGHS-1 and -2) each able to catalyze the committed step in PG formation-oxygenation of the omega 6 fatty acid arachidonic acid (AA) or the co3 fatty acid eicosapentaenoic acid. PGHSs, also known as cyclooxygenases (COXs), are products of different genes; typically, PGHS-1 is expressed constitutively, while PGHS-2 is expressed transiently. Each PGHS isoform subserves different biologies, and a central question is how this can occur. PGHS-1 displays negative substrate cooperativity. We posit that this restricts PGHS-1 to operating only at high AA concentrations when a bolus of PGs is required for a pulsatile, housekeeping event-something that could happen at any time during the cell cycle. Unlike PGHS-1, PGHS-2 can function at all substrate concentrations. We suggest that its normal function is to provide a slow, continuous synthesis of PGs during a 1-2 h period preceding cell differentiation or replication when AA levels are low and PGHS-2 is briefly present. In short, we hypothesize that regulation of PGHS-1 activity is kinetic while control of PGHS-2 activity resides in its expression. These ideas can explain how when the isoforms are co-expressed in cells, PGHS-2 can be active while PGHS-1 is latent. The kinetic properties of PGHS-1 permit its functioning in vitro only when AA or EPA levels reach > 1-2 uM. It is probably rare that cellular EPA levels become this high; moreover, EPA is a very poor substrate for PGHS-1. So we suggest that except at unusually high EPA/AA ratios, EPA does not function as a substrate for PGHS-1 in vivo. Some beneficial effects of dietary fish oil could relate to the inactivity of PGHS-1 with EPA. Specific Aim #1 will test our concepts about the differences in the abilities of PGHS-1 and PGHS-2 to oxygenate low vs. high concentrations of endogenous AA vs. EPA in cells. Fibroblasts expressing PGHS-1 or PGHS-2 and having different EPA/AA ratios in their phospholipids will be cultured. PGE2 and PGEj, synthesis will be measured with cells stimulated to mobilize low vs. high levels of endogenous substrates. To determine if PGHS-1 can oxygenate EPA in vivo, PGHS-2 null mice will be fed fish oil and urinary, EPA-derived PGs will be quantified. Specific Aim #2 will examine an unexplored aspect of the control of PGHS-2 expression-protein degradation. Compared to PGHS-1 degradation, PGHS-2 degradation is rapid (tj/2 ~ 2 h). We have identified a 27 amino acid instability element (27-IE) near the C-terminus of PGHS-2 that targets it to the ER-associated degradation system. We will define structural features of the 27-IE involved in its function and will phenotype a newly engineered PGHS-2 knock-in mouse having a non-functional 27-IE.

描述（由申请人提供）：我们研究的长期目标是了解前列腺素（PG）合成是如何调节的。有两种PGH脱氢酶（PGHS-1和-2），每种都能够催化PG形成中的关键步骤-ω 6脂肪酸花生四烯酸（AA）或ω 3脂肪酸二十碳五烯酸的氧化。PGHS，也称为环加氧酶（COX），是不同基因的产物;通常，PGHS-1组成型表达，而PGHS-2瞬时表达。每种PGHS同种型都适用于不同的生物学，核心问题是这是如何发生的。PGHS-1显示负底物协同效应。我们认为，这限制PGHS-1仅在高AA浓度下工作时，需要一团PGs的脉动，管家事件，这可能发生在细胞周期的任何时候。与PGHS-1不同，PGHS-2可以在所有底物浓度下发挥作用。我们认为，它的正常功能是提供一个缓慢的，连续的合成PG在1-2小时期间细胞分化或复制时，AA水平低，PGHS-2是短暂的。简而言之，我们假设PGHS-1活性的调节是动力学的，而PGHS-2活性的控制在于其表达。这些想法可以解释当同种型在细胞中共表达时，PGHS-2如何可以是活跃的，而PGHS-1是潜伏的。PGHS-1的动力学特性仅在AA或EPA水平达到> 1-2 μ M时才允许其在体外发挥功能。细胞EPA水平变得如此之高可能是罕见的;此外，EPA是PGHS-1的非常差的底物。因此，我们认为，除了在异常高的EPA/AA比例，EPA不作为PGHS-1在体内的底物。鱼油的某些有益作用可能与PGHS-1与EPA的失活有关。具体目标#1将测试我们关于PGHS-1和PGHS-2在细胞中抑制低浓度与高浓度内源性AA与EPA的能力差异的概念。将培养表达PGHS-1或PGHS-2并在其磷脂中具有不同EPA/AA比率的成纤维细胞。将用经刺激以动员低水平与高水平内源性底物的细胞测量PGE 2和PGE 3合成。为了确定PGHS-1是否可以在体内降解EPA，将给PGHS-2缺失小鼠喂食鱼油，并定量尿中EPA衍生的PG。具体目标#2将检查PGHS-2表达控制的未探索方面-蛋白质降解。与PGHS-1相比，PGHS-2的降解速度较快（tj/2 ~ 2 h）。我们已经确定了一个27个氨基酸的不稳定性元件（27-IE）的C-末端附近的PGHS-2的目标，它的ER相关的降解系统。我们将定义参与其功能的27-IE的结构特征，并将表型化具有非功能性27-IE的新工程化PGHS-2敲入小鼠。