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)合成是如何调节的。有两个PGH合成酶(PGHS-1和-2)，每一个都能够催化PG形成的关键步骤-氧化omega 6脂肪酸花生四烯酸(AA)或CO3脂肪酸二十碳五烯酸。PGHS，也称为环氧合酶(Coxs)，是不同基因的产物；通常，PGHS-1是组成性表达，而PGHS-2是瞬时表达。每一种PGHS亚型都伴随着不同的生物学，一个中心问题是这种情况是如何发生的。PGHS-1表现为负的底物协同作用。我们假设，这限制了PGHS-1仅在高AA浓度下工作，而在细胞周期的任何时候都可能发生这种情况，需要一剂PGs来处理脉动性的内务事件。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，它的正常功能是在细胞分化或复制前1-2小时内提供缓慢、连续的PGs合成，此时AA水平较低，且PGHS-2短暂存在。简而言之，我们假设PGHS-1活性的调节是动力学的，而PGHS-2活性的控制取决于其表达。这些想法可以解释为什么当这些亚型在细胞中共表达时，PGHS-2可以是活跃的，而PGHS-1是潜伏的。只有当AA或EPA水平达到1-2微米时，PGHS-1的动力学特性才允许其在体外发挥作用。细胞EPA水平变得如此高可能是罕见的；此外，EPA对PGHS-1来说是非常糟糕的底物。因此，我们认为，除非EPA/AA比值异常高，否则EPA在体内不能作为PGHS-1的底物。饲料中添加鱼油的一些有益效果可能与抑制PGHS-1与EPA的结合有关。具体目标#1将测试我们的概念，即PGHS-1和PGHS-2在细胞内氧化内源性AA和EPA的低浓度和高浓度的能力的差异。培养表达PGHS-1或PGHS-2且其磷脂中具有不同EPA/AA比例的成纤维细胞。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是否可以在体内氧化EPA，将给PGHS-2基因缺失的小鼠喂食鱼油和尿液，并对EPA来源的PGs进行量化。具体目标#2将研究PGHS-2表达控制的一个未知方面--蛋白质降解。与PGHS-1的降解相比，PGHS-2的降解速度更快(tj/2~2 h)。我们已经在PGHS-2的C末端附近发现了一个27个氨基酸的不稳定元件(27-IE)，它针对内质网相关的降解系统。我们将定义与其功能相关的27-IE的结构特征，并将表型为具有非功能27-IE的新工程PGHS-2敲入小鼠。

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.