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REGULATION OF THE PGH SYNTHASE GENES

PGH 合酶基因的调控

基本信息

批准号：
2684895
负责人：
David Lee DEWITT
金额：
$ 17.95万
依托单位：
MICHIGAN STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1988
资助国家：
美国
起止时间：
1988-07-01 至 2000-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2684895
关键词：
antiinflammatory agents biological models clone cells dexamethasone eicosanoid metabolism enzyme biosynthesis enzyme structure enzyme substrate complex glucocorticoids isozymes laboratory rabbit messenger RNA posttranscriptional RNA processing prostaglandin endoperoxide synthase protein signal sequence protein structure function transfection

项目摘要

Prostaglandin endoperoxide H(PGH) synthase (cyclooxygenase) is a central enzyme in the prostaglandin biosynthetic pathway and also the primary site of action of aspirin and other non-steroidal anti-inflammatory drugs (NSAIDs). Mammalian cells contain two related, but unique, forms of PGH synthase, referred to as PGH synthase-1 (PGHS-1) and PGH synthase-2 (PGHS02). These two enzymes appear to have specialized functions; PGHS-1 produces prostaglandins that act extracellularly s local hormones to coordinate short-term cellular responses to hormonal stimulation; while PGHS-2 produces prostaglandins that coordinate prolonged physiological events such as inflammation and ovulation. PGHS-2 produces prostaglandins that are directed into the nucleus where, we would predict, they also help regulate mitogenesis. The primary hypothesis that we propose to test in this application is that PGHS-1 and pGHS-2 form two separate prostaglandin biosynthetic pathways. Immunocytochemical localization by us indicates that PGHS-1 and PGHS-2 are located on different cellular membranes and produce prostaglandins that are shunted to different intracellular spaces. PGHS-1 is located predominantly in the endoplasmic reticulum and produces prostaglandins that are released into the cytoplasm where they presumably exit the cell to signal via extracellular receptors. PGHS-2 is located predominately on the nuclear membrane and releases prostaglandin on the nucleus and cytoplasm. How prostaglandin might signal in the nucleus is not known. Recently reported experiments indicate the PGHS-1 and PGHS-2 use separate arachidonate acid pools. To establish model systems with which to study the biochemical basis for segregation of the two prostaglandin synthase pathways, we are proposing to construct a set of 10 stably transfected cells which can inducibly express native and variously modified PGHS-1 and PGHS-2 proteins. These cell lines will be used to study the structure-function relationships that determine substate coupling, product channeling, and intracellular localization of PGHS-1 and PGHS-1 and PGHS-2 (Specific Aims 1-2), and also to examine the protein and mRNA sequences responsible for post-transcriptional regulation of PGHS-2 mRNA and protein stability (Specific Aims 3-4). If separate systems for prostaglandin synthesis do exist, with different mechanisms for arachidonate release and different effector pathways, these pathways may provide additional sites for therapeutic intervention of inflammation. in addition, a more complete understanding of the cellular roles for prostaglandin synthesis by PGHS-1 and PGHS-2 may help us to better understand the protective effects of NSAIDs on certain cancers.

前列腺素内源性过氧化氢(PGH)合成酶(环氧合酶)是一种前列腺素生物合成途径中的酶及其主要部位阿司匹林和其他非类固醇抗炎药的作用 (非甾体抗炎药)。哺乳动物细胞含有两种相关但独特的PGH 合酶，又称PGH合酶-1(PGHS-1)和PGH合酶-2 (PGHS02)。这两种酶似乎具有特殊的功能；PGHS-1 产生前列腺素，使S局部激素发挥细胞外作用协调细胞对激素刺激的短期反应； PGHS-2产生前列腺素来协调长时间的生理炎症和排卵等事件。PGHS-2产生前列腺素直接进入细胞核，我们可以预测，它们也有助于调节有丝分裂。我们建议测试的主要假设是这个应用是PGHS-1和pGHS-2形成两个独立的前列腺素生物合成途径。我们的免疫细胞化学定位表明 PGHS-1和PGHS-2位于不同的细胞膜上，产生前列腺素，分流到不同的细胞内空间。 PGHS-1主要定位于内质网，并产生前列腺素释放到细胞质中，在那里它们可能离开细胞，通过细胞外受体发出信号。PGHS-2卫星位于主要分布在核膜上，并在核膜上释放前列腺素胞核和细胞质。前列腺素在细胞核中的信号传递方式不知道。最近报道的实验表明，PGHS-1和PGHS-2 使用单独的花生四烯酸盐池。通过以下方式建立模型系统从而研究两者分离的生化基础。前列腺素合成酶通路，我们建议构建一组10 可诱导表达天然基因和多种基因的稳定表达细胞修饰的PGHS-1和PGHS-2蛋白。这些细胞系将被用于研究决定亚状态的结构-功能关系 PGHS-1和PGHS-1的偶联、产物通道和细胞内定位 PGHS-1和PGHS-2(特异性靶标1-2)，并检测蛋白质和参与PGHS-2转录后调控的mRNA序列信使核糖核酸和蛋白质稳定性(特定目标3-4)。如果单独的系统用于前列腺素的合成确实存在，有不同的机制花生四烯酸释放和不同的效应器途径，这些途径可以为炎症的治疗干预提供更多的场所。在……里面此外，还可以更全面地了解 PGHS-1和PGHS-2合成前列腺素可能有助于我们更好地了解非类固醇抗炎药对某些癌症的保护作用。