STRUCTURE/FUNCTION OF PROSTAGLANDIN H SYNTHASE 2

前列腺素，多不饱和脂肪酸的含氧代谢物家族 脂肪酸，已被认为在许多不同的 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。第一个承诺 在这些前列腺素的生物合成中的每一步，一个关键点 对这些强大的调解人水平的控制，是由 前列腺素H合成酶(PGHS)环氧合酶活性。 已发现两种PGHS亚型。最近的蛋白质和mRNA水平 已发现的第二种PGHS亚型，PGHS-2，在 静止的细胞，但在少数细胞中强烈而短暂地被诱导 按炎症介质分类。相比之下，PGHS-1在广泛的 各种静止的细胞，芽其水平仅被调节到有限 学位。很明显，这两种异构体具有不同的病理生理机制。 功能。新出现的概念是PGHS-2对前列腺素负责 合成对炎症至关重要，而PGHS-L为炎症产生前列腺素 内务管理功能。我们的长期目标是理解 PGHS-2的生化特性在其独特的功能中发挥作用 在活体内。具体目标是： L)确定了细胞差异调控的生化基础 PGHS-2合成前列腺素的研究。表达人PGHs的培养细胞 亚型和纯化的人类酶本身将被用来 评估催化控制的几个方面，包括对 过氧化氢活化剂，专用于脂肪酸底物结构， 和底物区隔。 2)表征了人PGHS-2的反应动力学，并进行了相关动力学研究 与人PGHS-L的差异在于特定蛋白质结构的差异。 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，并检查不含酪氨酸的作用 催化中的自由基。人类PGHS亚型的结构模型将 用于确定PGHS-2的结构特征，以供现场评估- 定向诱变。 3)对PGHS-2细胞内质网膜附着特性的研究 并与PGHS-1进行比较。膜附着的性质将 从特定多肽序列的可及性来评估 细胞质和内质网管腔，以及特定突变的影响 关于膜缔合。

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.