STRUCTURE/FUNCTION OF PROSTAGLANDIN H SYNTHASE 2

前列腺素类，多不饱和脂肪酸的含氧代谢产物家族， 脂肪酸，已被赋予重要的作用，在各种各样的 生理和病理过程包括炎症， 止血、动脉粥样硬化、血栓形成和中风。的第一个关键 这些前列腺素类的生物合成中的每一个步骤， 控制这些强大的介质的水平，是由催化剂， 前列腺素H合酶（PGHS）的环氧合酶活性。 目前已发现PGHS的两种亚型。 发现的第二种PGHS同种型PGHS-2，几乎检测不到， 静止细胞，但在少数细胞中强烈和短暂诱导 炎症介质的类型。相比之下，PGHS-1在广泛的 各种静止细胞，芽其水平被调制到仅有限 ℃下很明显，两种亚型具有不同的病理生理学差异。 功能协调发展的新出现的概念是PGHS-2负责前列腺素 合成对炎症至关重要，而PGHS-1产生前列腺素类， 内务职能。长期目标是了解 PGHS-2的生化特性决定了其独特的功能 in vivo.具体目标是： l）确定差异细胞控制的生物化学基础， PGHS-2合成前列腺素。表达人PGHs的培养细胞 同种型和纯化的人酶本身将用于 评估催化控制的几个方面，包括以下要求 氢过氧化物活化剂，对脂肪酸底物结构特异性， 和底物区室化。 2)对人PGHS-2的反应动力学进行表征， 与人PGHS-1的差异在于特定蛋白质结构差异。 纯化的人PGHS亚型将用于分析个体 环氧合酶和过氧化物酶反应步骤，以定量动力学 与抑制剂的相互作用，并检查酪氨酰自由的作用， 自由基在催化中的作用人PGHS同种型的结构模型将 用于确定PGHS-2的结构特征，以便按研究中心进行评估- 定向诱变 3)PGHS-2的内质网膜附着特征 并与PGHS-1进行比较。膜附着的性质将 可以从特定多肽片段的可及性来评估， 细胞质和内质网腔，以及特定突变的影响， 关于膜结合。

The prostanoids, a family of oxygenated metabolites of polyunsaturated fatty acids, have been ascribed important roles in a wide variety of physiological and pathological processes including inflammation, hemostasis, atherogenesis, thrombosis, and stroke. The first committed step in the biosynthesis of each of these prostanoids, a key point for control of the levels of these powerful mediators, is catalyzed by the cyclooxygenase activity of prostaglandin H synthase (PGHS). Two PGHS isoforms have been found. Protein and mRNA levels of the recently discovered second PGHS isoform, PGHS-2, are almost undetectable in quiescent cells, but are strongly and transiently induced in a few cell types by inflammatory mediators. In contrast, PGHS-1 is found in a wide variety of quiescent cells, bud its levels are modulated to only limited degree. It is clear that the two isoforms have distinct pathophysiological functions. The emerging concept has PGHS-2 responsible for prostanoid synthesis crucial to inflammation, whereas PGHS-l produces prostanoids for housekeeping functions. The long term goal is to understand the role that the biochemical characteristics of PGHS-2 play in its distinct functions in vivo. The specific aims are: l) Identify the biochemical basis for differential cellular control of prostanoid synthesis by PGHS-2. Cultured cells expressing the human PGHs isoforms and the purified human enzymes themselves will be used to evaluate several aspects of catalytic control, including requirements for hydroperoxide activator, specificity for fatty acid substrate structure, and substrate compartmentation. 2) Characterize the reaction kinetics of human PGHS-2, and relate kinetic differences with human PGHS-l to specific protein structural differences. Purified human PGHS isoforms will be used to analyze individual cyclooxygenase and peroxidase reaction steps, to quantitate the kinetics of interactions with inhibitors, and to examine the role of tyrosyl free radicals in catalysis. Structural models of the human PGHS isoforms will be used to identify structural features of PGHS-2 for evaluation by site- directed mutagenesis. 3) Characterize the endoplasmic reticulum membrane attachment of PGHS-2 and compare it with that of PGHS-1. The nature of membrane attachment will be evaluated from the accessibility of particular polypeptide segrnents to the cytoplasm and the ER lumen, and from the effects of specific mutations on membrane association.