Role of Protein Kinase C regulatory domains in modulating alcohol actions

描述(申请人提供)：酗酒和酗酒是导致死亡的主要原因(美国每年有75,000-100,000人死亡)，在美国每年造成1840亿美元的经济负担。明确靶点(S)并阐明其作用的分子机制是有效干预的必要条件。这项建议的目的是确定酒精在脑细胞内信号转导途径中发挥作用的分子机制。众所周知，酒精会改变蛋白激酶Cs(PKC)的表达和活性，PKC是一种主要在大脑中表达的激酶家族。与野生型小鼠相比，PKC epsilon基因敲除小鼠的酒精消耗量显著减少，对乙醇的耐受性增加，而PKC Gamma基因敲除小鼠的酒精消耗量显著增加，耐受性降低。在调节GABAA(3-氨基丁酸)受体的反应中，PKCβ和PKC3基因敲除也表现出相反的特性。GABAA是脑内的一种配体门控离子通道，被认为是酒精的重要靶点。在结构上，PKC3在N端有其调控的C1结构域(C1a和C1B的组合)，紧随其后的是调控的C2结构域。另一方面，PKC？的C2结构域在N端，紧随其后的是C1域。Epsilon和Gamma的C1a和C1B亚域在配体结合特性方面存在显著差异。需要检验的中心假设是，PKC和PKC3调节域的结构和配体结合的差异是它们对酒精的不同行为反应的原因。这项建议的第一个目的是确定酒精如何调节PKC和PKC3的活性，以及在它们的调节域中是否存在酒精结合部位。为了确定醇的结合部位，我们将使用新型的醇的光活性二氮杂环类似物来光标记PKC结构域/亚结构域，然后用质谱仪对标记的残基进行鉴定。第二个目的是通过在PKC和PKC3之间交换调节结构域/亚结构域来产生PKC嵌合体，并表征它们的酒精结合特性。这将通过测量酒精对嵌合蛋白活性的影响来实现。第三个目的是通过电生理测量GABAA电流来研究酒精对GABAA-PKC相互作用的影响。这项建议将加深我们目前对酒精作用的分子机制的理解，并将为设计治疗酒精成瘾的新疗法提供策略。 与公共健康相关：尽管与酒精有关的死亡人数巨大(美国每年75000-100,000人)，社会成本(美国每年1,840亿美元)很少，但治疗与酒精有关的疾病的药物很少。为了开发新的药物和有效的预防，有必要在分子水平上确定其作用的靶点和机制。本研究的意义在于确定信号转导蛋白在调节酒精行为中的作用。这项研究将增进对酒精成瘾药物开发的认识。

DESCRIPTION (provided by applicant): Alcoholism and alcohol abuse is a major cause of death (annually 75,000-100,000 deaths in the US) with an economic burden of 184 billion dollars/year in the US. Defining the target(s) and elucidating the molecular mechanism of its action is needed for effective intervention. The objective of this proposal is to define the molecular mechanisms by which alcohols exert their action on intracellular signal transduction pathways in brain. Alcohols are known to alter the expression and activity of Protein Kinase Cs (PKC), a family of kinases mainly expressed in the brain. While PKC epsilon knock-outs showed significant decrease in alcohol consumption and increase in tolerance to ethanol compared to wild type mice, the PKC gamma knock-outs showed significant increase in alcohol consumption and decrease in tolerance. PKC¿ and PKC3 knock-outs also showed opposite properties in regulating responses to GABAA (3-amino butyric acid) receptors. GABAA is a ligand-gated ion channel in brain and believed to be an important target of alcohol. Structurally, PKC3 has its regulatory C1 domain (combination of C1A and C1B) at the N terminus followed by the regulatory C2 domain. On the other hand, PKC¿ has its C2 domain at the N terminus followed by the C1 domain. The C1A and C1B subdomains of epsilon and gamma differ significantly in terms of their ligand binding properties. The central hypothesis to be tested is that the structural and ligand binding differences in the regulatory domains of PKC¿ and PKC3 are responsible for their differential behavioral response to alcohol. The first aim of this proposal is to determine how alcohols regulate PKC¿ and PKC3 activity and if there is an alcohol binding site in their regulatory domains. In order to identify alcohol binding site we will use novel photoactive diazirine analog of alcohols to photolabel the PKC domain/subdomains followed by identification of the labeled residues by mass spectrometry. The second aim is to generate PKC chimeras by swapping the regulatory domains/subdomains between PKC¿ and PKC3 and characterize their alcohol binding properties. This will be achieved by measuring the effect of alcohols on the activities of the chimeric proteins. The third aim is to investigate the effect of alcohols on GABAA-PKC interactions by measuring GABAA current electrophysiologically. The proposal will enhance our current understanding on the molecular mechanism of alcohol action and will provide strategy for designing new therapeutics against alcohol addiction. PUBLIC HEALTH RELEVANCE: Despite huge number of alcohol related deaths (annually 75000-100,000 in the US) and cost to society ($184 billion/yr in the US) very few medications are available for treating alcohol related diseases. To develop new medications and effective prevention it is necessary to define the target and mechanism of its action at the molecular level. The significance of the present study is to establish the role of a signal transducing protein in regulating alcohol actions. This study will enhance the knowledge in developing medication for alcohol addiction.