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 前列腺素内源性过氧化物合成酶-1和-2(PGHS-1和PGHS-2) (也称为环氧合酶；COX-1和COX-2)。PGHS已经被 被广泛研究，因为它们在 前列腺素的合成，也是因为它们是 非类固醇抗炎药(NSAID)，如阿司匹林和 布洛芬。阿司匹林和其他非甾体抗炎药也能有效地减少 人类结肠癌的发病率，以及在抑制肿瘤形成方面的作用 结肠癌动物模型，表明前列腺素是 扩散和/或转化的重要调节者。然而， 非甾体抗炎药在炎症和癌症中的保护作用似乎是 是通过抑制PGHS-2而不是PGHS-1介导的。为了更好地 了解PGHS-1和PGHS-2的差异信号机制， 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定在脂质双层内。我们的Main 假说是这两种物质的膜相互作用的差异 同工酶影响它们的生物信号特性。我们的首要目标是 将描述功能性PGHS的结构特征 膜结合结构域，并确定是否存在差异 PGHS-1和PGHS-2的膜结合结构域序列发生改变 在膜内两种同工酶的方向上，这可能与 它们独特的生物学特性。我们的首要目标是 优化先前开发的用于Active重建的方法 PGHS-1和PGHS-2转化为脂泡。重建实验将 使我们可以用来确定PGHS-1和PGHS-2的膜形貌 三种不同的生化和物理方法：(A)特定地点 膜相关蛋白序列的标记；(B)EPR波谱 和(C)中子衍射散射分析。 氢化洗涤剂中的PGHS-1和PGHS-2的结晶。

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.