STRUCTURE/FUNCTION OF PROSTAGLANDIN H SYNTHASE-2

描述(申请人提供)：前列腺素H合成酶(PGHS)的环氧合酶活性催化前列腺素生物合成的第一步，前列腺素是一组对许多病理生理过程非常重要的生物活性脂类，包括炎症、血管、胃和肾功能、生殖和肿瘤发生。PGHS是髓过氧化物酶家族的一员，目前已知有两种亚型：PGHS-1通常被认为是结构性的，具有管家功能；PGHS-2在参与炎症和增殖过程的许多细胞中都能被细胞因子强烈诱导。除了对PGHS-1和-2基因表达的调控外，细胞前列腺素的合成也在环氧合酶的催化水平上受到严格的调控，对这两种PGHS亚型的催化调控相当不同。与PGHS-1环氧合酶相比，PGHS-2环氧合酶对氢过氧化氢激活剂的需求要低得多。这种副产物反馈激活的差异为通过细胞过氧化物酶对过氧化氢水平的抑制作用而对环氧合酶催化进行差异化控制提供了简单的生物化学基础。过氧化氢激活剂被用来在环氧合酶活性部位形成酪氨酸自由基；该自由基在PGHS-2中形成得更快，比在PGHS-1中更稳定。这个项目的总体目标是在分子水平上了解PGHS亚型对催化的调节。动力学、光谱和结构研究将对两种PGHS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，以实现以下特定目的：1)表征控制酪氨酸自由基的形成、稳定性和破坏性副反应的PGHS-2的结构特征，并确定PGHS-2中环氧合酶激活效率较高的结构基础；2)表征膜环境对PGHS-I和PGHS-2中的PGG2通道以及对磷脂过氧化氢谷胱甘肽过氧化物酶(HGPx)和胞浆谷胱甘肽过氧化物酶(GPx)的抑制作用；3)以鲑鱼PGHS亚型和植物病原菌诱导加氧酶(PiOX)为模型，评价哺乳动物PGHS反应机制和催化调控方案的共性。

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.