MONOTOPIC MEMBRANE ANCHORS IN PGHS SYNTHASES 1 AND 2

这项资助的主要重点是研究膜的相互作用， 前列腺素内过氧化物酶-1和-2（PGHS-1和PGHS-2） (also称为环氧合酶;考克斯-1和考克斯-2）。PGHS已经 广泛研究，因为他们的基本和监管作用， 前列腺素的合成，也因为它们是作用的网站， 非甾体抗炎药（NSAID），如阿司匹林， 布洛芬阿司匹林和其他非甾体抗炎药也有效地减少了 结肠癌的发病率，并在抑制肿瘤的形成， 结肠癌的动物模型，这表明， 增殖和/或转化的重要调节剂。但 非甾体抗炎药的保护作用，无论是在炎症和癌症，似乎 通过抑制PGHS-2而不是PGHS-1介导。更好地 了解PGHS-1和PGHS-2的差异信号传导机制， 我们将研究它们与膜的物理相互作用。这些 同工酶不包含跨膜序列，而是具有四个 短的连续的两亲性螺旋，或膜结合结构域， 被认为是将这些蛋白质锚在脂质双层内。我们的主要 假设这两种细胞膜相互作用的差异 同工酶影响它们的生物信号传导特性。我们的首要目标 将是表征功能性PGHS的结构特征 膜结合结构域，并确定是否差异， PGHS-1和PGHS-2的膜结合结构域序列导致 在膜内两种同工酶的取向中， 它们独特的生物学特性我们的首要目标是 优化先前开发的活性物质重构方法 PGHS-1和PGHS-2进入脂质囊泡。重组实验将 使我们能够确定PGHS-1和PGHS-2的膜形貌， 三种不同的生物化学和物理方法：（a）特定地点 膜相关蛋白序列的标记;（B） 自旋标记PGHS;和（c）中子衍射散射分析 PGHS-1和PGHS-2在氘代洗涤剂中的晶体。

The major focus of this grant is to examine the membrane interactions of the prostaglandin endoperoxide synthases-1 and -2 (PGHS-1 and PGHS-2) (also called cyclooxygenases; COX-1 and COX-2). The PGHSs have been studied extensively because of their essential and regulatory role in prostaglandin synthesis and also because they are the site of action for non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin and ibuprofen. Aspirin and other NSAIDs have also been effective in reducing the incidence of colon cancer in man, and in inhibiting tumor formation in animal models of colon cancer, suggesting that prostaglandins are important regulators of proliferation and/or transformation. However, the protective effects of NSAIDs, both in inflammation and cancer, appear to be mediated through inhibition of PGHS-2 and not PGHS-1. To better understand the mechanism for differential signaling by PGHS-1 and PGHS-2, we will examine their physical interactions with membranes. These isoenzymes do not contain trans-membrane sequences, but instead have four short contiguous amphipathic helices, or membrane binding domains, that are thought to anchor these proteins within the lipid bilayer. Our main hypothesis is that differences in the membrane interaction of these two isozymes affects their biological signaling properties. Our primary goal will be to characterize the structural features of a functional PGHS membrane binding domain, and to determine whether differences in the membrane binding domain sequences of PGHS-1 and PGHS-2 result in changes in the orientation of the two isozymes within membranes that may relate to their unique biological properties. Our first objective will be to optimize previously developed methods for the reconstitution of active PGHS-1 and PGHS-2 into lipid vesicles. Reconstitution experiments will allow us to determine the membrane topography of PGHS-1 and PGHS-2 using three different biochemical and physical methods: (a) site specific labeling of membrane associated protein sequences; (b) EPR spectroscopy of spin-labeled PGHSs; and (c) neutron diffraction scattering analysis of crystals of the PGHS-1 and PGHS-2 in deuterated detergents.