The Molecular Basis of Alcohol's Actions

描述(由申请者提供)：该项目的长期目标是了解酒精作用的分子机制。由于体积小，缺乏化学特性，酒精通常被认为是一种相对非特异性的药物。已经确定了许多细胞因素，这些因素有助于调节细胞对乙醇的反应，最终导致成瘾的发展。然而，很明显，酒精并不与所有这些蛋白质相互作用，但实际上对有限数量的受体、离子通道和其他信号分子显示出相对较高的特异性。这导致了一种假设，即在许多情况下，酒精通过与这些蛋白质中的特定位置结合来导致蛋白质结构和功能的变化。然而，人们对酒精结合部位的组成和酒精敏感性的影响知之甚少。此前，我们在果蝇蛋白LUSH中发现了一个特定的酒精结合位点。对该蛋白质的结构和生化研究发现，需要多个氢键残基的协同作用才能形成乙醇结合部位。在这项工作中，我们将重点确定酒精与蛋白激酶C(PKC)家族酶的调节结构域之间的相互作用。几种PKC蛋白的异构体在调节对乙醇的反应中起着中心作用。此外，PKC的几种异构体对酒精直接敏感，这表明乙醇可以调节亚细胞定位和激活所需的重要辅助因子的结合。我们将使用结构生物学方法来确定酒精如何与这些结构域相互作用，以及这些相互作用如何导致结构和动力学的变化，从而改变PKC结构域与辅助因子相互作用的能力。我们已经收集了初步数据，确定了酒精和PKC-α之间的特定相互作用。最后，我们建议研究酒精与甘氨酸受体跨膜结构域之间的相互作用。甘氨酸受体是酒精在中枢神经系统中的主要靶点之一，大量研究已经开发出酒精如何影响这一受体功能的模型。我们的研究将确定哪些残基对结合酒精至关重要，已知的突变如何可能改变其对酒精的敏感性与公共卫生相关了解乙醇如何作用于调节蛋白质结构和功能是一个与人类健康相关的基本问题。确定酒精在蛋白质中的作用部位，以及这些部位上的氨基酸如何促进乙醇敏感性，将有助于更好地理解酒精的作用机制。这可能会使未来开发新的方法来防止酒精行为，并识别与酒精中毒易感性有关的遗传标记。

DESCRIPTION (provided by applicant): The long term goal of this project is to understand the molecular mechanisms of alcohol's actions. Due to its small size and lack of chemical features, alcohol is often considered to be a relatively non-specific drug. Many cellular factors have been identified that contribute to regulating cellular responses to ethanol that ultimately lead to the development of addiction. However, it is clear that alcohol does not interact with all these proteins, but in fact shows a relatively high degree of specificity for a limited number of receptors, ion channels and other signaling molecules. This led to the hypothesis that in many cases alcohol acts by binding to specific sites in these proteins to bring about changes in protein structure and function. However, there is very little knowledge about what makes up an alcohol binding site and contribute to alcohol sensitivity. Previously, we identified a specific alcohol binding site in the Drosophila protein LUSH. Structural and biochemical studies of this protein led to the discovery that concerted interactions from multiple hydrogen bonding resides were required to form an ethanol binding site. In this work we will focus on determining the interactions between alcohol and regulatory domains of Protein Kinase C (PKC) family of enzymes. Several isoforms of PKC proteins play a central in regulating responses to ethanol. Further, several isoforms of PKC have been shown to be directly sensitive to alcohol and it is suggested that ethanol can modulate binding of important cofactors required for sub-cellular localization and activation. We will use structural biology methods to determine how alcohol interacts with these domains, and how these interactions lead to changes in structure and dynamics that could alter the ability of PKC domains to interact with co-factors. We have already collected preliminary data that have identified a specific interaction between alcohol and PKC-alpha. Finally we propose to investigate the interactions between alcohol and the transmembrane domains of the glycine receptor. Glycine receptors are one of the primary targets of alcohol in the central nervous systems, and a wealth of studies have developed a model for how alcohol may affect the function of this receptor. Our studies will determine what residues are critical for binding alcohol, an how the known mutations are likely to its modify alcohol sensitivity PUBLIC HEALTH RELEVANCE Understanding how ethanol acts to modulate protein structure and function is a fundamental problem relevant to human health. Defining the sites of alcohol actions in proteins, and how amino acids in these sites contribute to ethanol sensitivity, will provide a greater understanding of the mechanism of alcohol's actions. This may allow future development of novel approaches to prevent alcohols actions, and the identification of genetic markers associated with a predisposition to alcoholism.