Catalysis by Prostaglandin Endoperoxide H Synthases

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）合成是如何被调节的。有两种PGH合成酶（PGHS-1和pghs -2）都能够催化PG形成的特定步骤- omega - 6脂肪酸花生四烯酸（AA）或co3脂肪酸二十碳五烯酸的氧化。pghs，也被称为环氧化酶（cox），是不同基因的产物；通常，PGHS-1是本构性表达，而PGHS-2是瞬时表达。每个PGHS异构体都服务于不同的生物学，一个核心问题是这是如何发生的。PGHS-1表现出负的底物协同性。我们假设，这限制了PGHS-1仅在高AA浓度下工作，当脉动性、内务性事件（可能在细胞周期的任何时候发生）需要大量PGs时。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，当AA水平较低且PGHS-2短暂存在时，其正常功能是在细胞分化或复制前的1-2小时内提供缓慢、连续的pg合成。总之，我们假设PGHS-1活性的调节是动态的，而PGHS-2活性的控制则在于其表达。这些想法可以解释为什么当同种异构体在细胞中共表达时，PGHS-2可以活跃，而PGHS-1是潜伏的。PGHS-1的动力学性质允许其在体外仅当AA或EPA水平达到1-2 μ m时才能发挥作用。细胞内的EPA水平如此之高可能是罕见的；此外，EPA对于PGHS-1来说是一个很差的底物。因此，我们认为，除非在异常高的EPA/AA比率下，EPA在体内不会作为PGHS-1的底物起作用。膳食鱼油的一些有益作用可能与PGHS-1在EPA作用下不活跃有关。特异性目标#1将测试我们关于PGHS-1和PGHS-2在细胞中氧化低浓度与高浓度内源性AA和EPA的能力差异的概念。培养表达PGHS-1或PGHS-2且磷脂中EPA/AA比例不同的成纤维细胞。PGE2和PGEj的合成将通过刺激细胞动员低水平和高水平的内源性底物来测量。为了确定PGHS-1是否能在体内氧化EPA，我们给PGHS-2缺失的小鼠喂食鱼油，并对尿液中EPA衍生的PGs进行量化。特异性目标#2将研究控制PGHS-2表达蛋白降解的一个未探索的方面。与PGHS-1降解相比，PGHS-2降解速度较快（tj/2 ~ 2h）。我们在PGHS-2的c端附近发现了一个27个氨基酸的不稳定元件（27- ie），将其靶向于er相关的降解系统。我们将定义与其功能相关的27-IE的结构特征，并对具有非功能27-IE的新工程PGHS-2敲入小鼠进行表型分析。

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.