Proteomics of Nicotinic Receptor Complexes

烟碱受体复合物的蛋白质组学

• 批准号: 7390196
• 负责人: Rene Anand
• 金额: $ 16.4万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-03-15 至 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7390196
• 关键词: Proteomics; Nicotinic; Receptor; Complexes

DESCRIPTION (provided by applicant): Nicotinic acetylcholine receptors (AChRs) mediate the actions of nicotine in tobacco in the central nervous system (CMS). We wish to understand the molecular mechanisms by which the repetitive activation of AChRs by nicotine initiates and sustains the molecular and cellular changes of the CMS that occur during tobacco addiction. Our central hypothesis is that neuronal AChRs are ion channels that are associated with distinct sets of cytosolic proteins of different functions. It is likely that these proteins, and the processes they regulate, participate in the changing the density, functional organization, and properties of AChRs following their repetitive activation by nicotine. These changes in AChRs then lead to downstream adaptive changes in neural networks within which they are expressed (e.g. mesocorticolimbic dopamine system) and thus sustain addiction to nicotine. The main objective of this proposal is to identify cytosolic proteins associated both directly and indirectly with the alphas and alpha? AChR subunits, because they are representative subunits of two major AChR subtypes expressed in neurons. We will to accomplish this by combining two approaches: 1) a new proteomic approach using soluble fusion proteins of tagged AChR subunit cytoplasmic loops, heterologously expressed in neural cells, to affinity purify associated proteins, which will then be identified by mass spectrometry; and 2) by performing yeast two-hybrid screens of cell line-derived, and thus enriched, cDNA libraries. We are applying for a R21 because a part of the proposed study adapts an innovative, but high risk proteomic approach, which has only been used to purify soluble protein complexes, to identify protein complexes associated with integral membrane proteins. We hypothesize that these proteins or the pathways in which they function could be additionally targeted with high-specificity by the development of drugs that disrupt their interactions with specific sequences in this domain to modulate the biogenesis of AChRs, and thus specific physiological effectors of nicotine's action in the CMS. Nicotinic receptors have been shown to be affected in many diseases including schizophrenia and tobacco addiction. The proposed work will clarify what other novel proteins regulate their function in brain nerve cells and thus provide new targets for therapeutic manipulation for the treatment of these neurolgical diseases.

描述（由申请人提供）：烟碱乙酰胆碱受体（ACHR）介导了中枢神经系统（CMS）中烟碱的作用。我们希望理解尼古丁对ACHR的重复激活的分子机制，可以启动并维持烟草成瘾过程中CMS的分子和细胞变化。我们的中心假设是神经元ACHR是与不同功能的胞质蛋白集相关的离子通道。这些蛋白质及其调节的过程很可能参与了ACHR被尼古丁重复激活后ACHR的密度，功能组织和特性。然后，ACHR中的这些变化导致其表达的神经网络的下游自适应变化（例如，中皮质胶质糖多巴胺系统），从而维持对尼古丁的成瘾。该提案的主要目的是直接和间接与Alpha和Alpha直接和间接相关的胞质蛋白？ ACHR亚基，因为它们是在神经元中表达的两个主要ACHR亚型的代表性亚基。我们将通过结合两种方法来实现这一目标：1）一种使用标记为ACHR亚基的可溶性融合蛋白的新蛋白质组学方法，在神经细胞中异质表达，与亲和力纯化相关的蛋白质，然后通过质谱法鉴定出相关的相关蛋白； 2）通过执行细胞系衍生的酵母两杂交筛选，并因此富集cDNA库。我们正在申请R21，因为拟议研究的一部分适应了一种创新但高风险的蛋白质组学方法，该方法仅用于净化可溶性蛋白质复合物，以鉴定与整合性膜蛋白相关的蛋白质复合物。我们假设这些蛋白质或它们发挥作用的途径可以通过开发药物的开发来替代高特异性，从而破坏了它们与该结构域中特定序列的相互作用以调节ACHR的生物发生，从而破坏了nicotine在CMS中尼古丁作用的特定生理效应因子。烟碱受体已被证明在许多疾病中受到影响，包括精神分裂症和烟草成瘾。拟议的工作将阐明其他新型蛋白质在脑神经细胞中的功能，从而为治疗这些神经性疾病治疗的治疗操作提供了新的靶标。