Dissecting the Cytoplasmic Interactome of the Alpha7 nACHR Subtype

描述(申请人提供)：这项研究的目标是发现A7烟碱型乙酰胆碱受体(NAChR)亚型的细胞质相互作用组，以及这种相互作用组如何因慢性尼古丁暴露而改变。虽然对nAChR的许多方面已经有了很好的研究，但这些受体在细胞内的蛋白相互作用仍不清楚。了解这些细胞质蛋白质的相互作用将有助于更好地理解这种受体的生理和病理。A7nAChR亚型与许多病理疾病有关，包括癫痫障碍、阿尔茨海默病、弥漫性路易体痴呆和尼古丁成瘾。了解尼古丁如何影响天然的A7 nAChR互动组，可以确定戒烟治疗的药物靶点，并为尼古丁的有益方面提供洞察，如增强认知和预防阿尔茨海默病。许多与慢性吸烟有关的病理情况往往是内在性的，随着时间的推移，患者的生活质量会急剧下降。戒烟是防止这些病症变得暴发性的关键。该项目的具体目标是利用特定毒素和尖端质谱学分离受体，以产生A7 nAChR相互作用组的体内图像。实验模型将是野生型小鼠，而A7nAChR基因敲除小鼠将作为对照。本项目的第一个具体目标是确定A7nAChRs的天然相互作用组。从野生型和A7基因敲除小鼠中提取的整个大脑将被均质。A7nAChR亚型将从含有a-银环蛇毒素(BGTX)的匀浆中纯化。A7nAChR和任何与其结合的相互作用蛋白可以通过氨甲酰胆碱洗脱从BGTX中释放出来。从BGTX分离得到的蛋白质将被蛋白水解酶降解，并用对大分子浓度的分子敏感的质谱仪进行分析。我们可以使用的质谱计是极其专业的仪器，具有最高的质量精度和分辨率。这些仪器还可以执行串联质谱仪，这是一种产生多肽氨基酸序列信息的技术，有助于准确识别蛋白质。串联质谱仪也可以用来评估多肽的磷酸化状态。第二个具体目标将是确定慢性尼古丁暴露如何影响A7 nAChR互动组。在特定目标1中使用的方案将在暴露于尼古丁5个月的野生型和A7nAChR基因敲除小鼠上重复使用。从尼古丁接触组小鼠收集的相互作用组数据将与未接触尼古丁组小鼠进行比较，以确定是否存在任何差异。

DESCRIPTION (provided by applicant): The goals of this study are to discover the cytoplasmic interactome of the a7 nicotinic acetylcholine receptor (nAChR) subtype and how this interactome is altered by chronic nicotine exposure. Although many aspects of the nAChR have been well studied, the intracellular protein interactions of these receptors are still unknown. Learning about these cytoplasmic protein interactions would generate a better understanding of this receptor's physiology and pathology. The a7 nAChR subtype has been linked to many pathological conditions including seizure disorders, Alzheimer's disease, diffuse lewy body dementia, and nicotine addiction. Understanding how the native a7 nAChR interactome is influenced by nicotine could identify drug targets for smoking cessation therapy as well as provide insight into nicotine's beneficial aspects such as cognitive enhancement and protection against Alzheimer's disease. The numerous pathological conditions linked to chronic smoking are frequently insidioust and over time drastically reduce a patient's quality of life. Smoking cessation is key to preventing these pathologies 'from becoming fulminant. The specific aims of this project employ the isolation of receptors by a specific toxin and cutting edge mass spectrometry to generate an in vivo picture of the a7 nAChR interactome. The experimental models will be wildtype mice and a7 nAChR knockout mice will function as a control. The first specific aim of this project is to identify the native interactome of a7 nAChRs. Whole brains extracted from wildtype and a7 knockout mice will be homogenized. a7 nAChR subtypes will be purified from the homogenate with a-bungarotoxin (Bgtx) beads. The a7 nAChR and any interacting proteins bound to it can be released from the Bgtx by carbachol elution. The proteins derived from Bgtx isolation will be degraded by proteolytic enzymes and analyzed by mass spectrometers sensitive to molecules at attomolar concentrations. The mass spectrometers available to us are extremely specialized instruments with the highest mass accuracy and resolution available. These instruments can also perform tandem mass spectrometry, a technique that generates peptide amino acid sequence information to facilitate accurate protein identification. Tandem mass spectrometry can also be used to asses the phosphorylation status of peptides. The second specific aim will be to determine how the a7 nAChR interactome is influenced by chronic nicotine exposure. The protocol used in specific aim 1 will be repeated on wildtype and a7 nAChR knockouts mice that have been exposed to nicotine for 5 months. The interactome data gathered from the nicotine exposed mice will be compared to the unexposed mice to determine if any difference exists.