STRUCTURE/FUNCTION OF PROSTAGLANDIN H SYNTHASE-2

描述（由申请人提供）：前列腺素H合成酶（PGHS）的环加氧酶活性催化了前列腺素生物合成的第一步，前列腺素是一组有效的生物活性脂质，对许多病理生理过程很重要，包括炎症、血管、胃和肾脏功能、生殖和肿瘤发生。PGHS是髓过氧化物酶家族的成员，已知有两种PGHS亚型：PGHS-1通常被认为是组成型的，具有管家功能；PGHS-2在许多参与炎症和增殖过程的细胞中被细胞因子强烈诱导。除了控制PGHS-1和-2基因的表达外，细胞前列腺素的合成在环加氧酶的催化水平上也受到严格的调控，对两种PGHS异构体的催化控制非常明显。与PGHS-1环加氧酶相比，PGHS-2环加氧酶对过氧化氢活化剂的需求要低得多。这种反馈激活副产物的差异为通过抑制细胞过氧化物酶对过氧化物水平的作用来控制环加氧酶催化的细胞差异提供了简单的生化基础。过氧化活化剂用于在环加氧酶活性位点形成酪氨酸自由基；这种自由基在PGHS-2中比在PGHS-1中形成得更快更稳定。本项目的总体目标是在分子水平上了解PGHS异构体对催化作用的调控。将对PGHS的两种异构体、另一种来自髓过氧化物酶家族的脂肪酸加氧酶和靶向突变蛋白进行动力学、光谱和结构研究，以实现以下具体目标：1)表征PGHS-2控制酪氨酸自由基形成、稳定性和破坏性副反应的结构特征，并确定PGHS-2中较高的环加氧酶激活效率的结构基础；2)表征膜环境对pghs - 1和pghs -2中PGG2通道的影响以及对磷脂氢过氧化物谷胱甘肽过氧化物酶（HGPx）和胞质谷胱甘肽过氧化物酶（cGPx）抑制作用的影响；3)以鳟鱼PGHS异构体和植物病原体诱导加氧酶（PIOX）为模型，评价哺乳动物PGHS反应机理和催化调控方案的通用性。

DESCRIPTION (provided by applicant): The cyclooxygenase activity of prostaglandin H synthase (PGHS) catalyzes the first committed step in biosynthesis of the prostaglandins, a group of potent bioactive lipids important to many pathophysiological processes, including inflammation, vascular, gastric and renal function, reproduction, and tumorigenesis. PGHS is a member of the myeloperoxidase family and two PGHS isoforms are known: PGHS-1 is generally regarded as constitutive, with housekeeping functions; PGHS-2 is strongly inducible by cytokines in many cells involved in inflammatory and proliferative processes. Besides controls on PGHS-1 and -2 gene expression, cellular prostaglandin synthesis is also tightly regulated at the cyclooxygenase catalytic level, with quite distinct catalytic controls for the two PGHS isoforms. PGHS-2 cyclooxygenase has a much lower hydroperoxide activator requirement than PGHS-1 cyclooxygenase. This difference in feedback activation by product provides a simple biochemical basis for the differential cellular control of cyclooxygenase catalysis via suppressive actions of cellular peroxidases on peroxide levels. Peroxide activator is used to form a tyrosyl radical in the cyclooxygenase active site; this radical forms faster and is more stable in PGHS-2 than in PGHS-1. The general goal of this project is to understand the regulation of catalysis by the PGHS isoforms at a molecular level. Kinetic, spectroscopic, and structural studies will be undertaken with the two PGHS isoforms, another fatty acid oxygenase from the myeloperoxidase family, and targeted mutant proteins to achieve the following specific aims: 1) Characterize the PGHS-2 structural features governing tyrosyl radical formation, stability, and destructive side reactions and identify the structural basis for the higher cyclooxygenase activation efficiency in PGHS-2; 2) Characterize the effects of a membrane environment on PGG2 channeling in PGHS-I and -2 and on suppressive actions of phospholipid hydroperoxide glutathione peroxidase (HGPx) and cytosolic glutathione peroxidase (cGPx); and 3) Evaluate the generality of mammalian PGHS reaction mechanisms and catalytic regulation schemes using trout PGHS isoforms and plant pathogen-induced oxygenase (PIOX) as models.