STRUCTURE/FUNCTION OF PROSTAGLANDIN H SYNTHASE-2

描述（由申请人提供）：前列腺素H合酶（PGHS）的环氧合酶活性催化前列腺素生物合成的第一个关键步骤，前列腺素是一组对许多病理生理过程（包括炎症、血管、胃和肾功能、生殖和肿瘤发生）重要的有效生物活性脂质。 PGHS是髓过氧化物酶家族的成员，已知有两种PGHS同种型：PGHS-1通常被认为是组成型的，具有管家功能; PGHS-2在参与炎症和增殖过程的许多细胞中被细胞因子强烈诱导。除了对PGHS-1和PGHS-2基因表达的控制外，细胞前列腺素合成也在环氧合酶催化水平上受到严格调节，对两种PGHS亚型具有非常不同的催化控制。PGHS-2环加氧酶比PGHS-1环加氧酶具有低得多的氢过氧化物活化剂需求。反馈激活产物的这种差异为通过细胞过氧化物酶对过氧化物水平的抑制作用对环加氧酶催化的差异细胞控制提供了简单的生化基础。过氧化物活化剂用于在环氧合酶活性位点形成酪氨酰自由基;该自由基在PGHS-2中比在PGHS-1中形成更快且更稳定。本项目的总体目标是了解PGHS异构体在分子水平上对催化的调节。本研究将对PGHS-2的两种异构体、髓过氧化物酶家族的另一种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中控制酪氨酰自由基形成、稳定性和破坏性副反应的结构特征，并鉴定PGHS-2中较高的环氧合酶激活效率的结构基础; 2）表征膜环境对PGHS-1和PGHS-2中的PGG 2通道以及对磷脂氢过氧化物谷胱甘肽过氧化物酶（HGPx）和细胞溶质谷胱甘肽过氧化物酶（cGPx）的抑制作用的影响;和3）使用鳟鱼PGHS同种型和植物病原体诱导的加氧酶（PIOX）作为模型，评估哺乳动物PGHS反应机制和催化调节方案的一般性。

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.