Analysis of the Dynamic Synaptic Proteome

描述(由申请人提供)：突触是动态的，与它们的突触传输的大小、它们的形态结构和它们的蛋白质成分有关。突触蛋白质组的动态变化受蛋白质合成、降解、分布和翻译后修饰的影响，影响突触的功能和突触的可塑性。尽管人们认识到突触的生物学重要性 蛋白质动力学，即丰富和鉴定动态突触蛋白质组的方法，目前还不是神经科学研究的组成部分。大量在原核生物和非神经细胞中进行的研究表明，利用非规范氨基酸掺入新合成的蛋白质中，然后生物正交点击化学用生物素等探针标记非规范氨基酸标记的蛋白质，可用于新合成蛋白质的纯化和鉴定。我们建议在神经细胞培养中以及在正常大鼠和非洲爪哇蝌蚪的视觉系统中优化使用非规范氨基酸掺入和生物正交点击化学的实验，以证明代谢标记用于识别和定量突触蛋白质组动态成分的可行性。这些实验旨在为比较不同条件和不同实验系统中突触蛋白质的合成提供原则性证据。 与公共健康相关：新合成的突触蛋白调节突触功能和可塑性。我们建议改进使用非规范氨基酸的代谢标记来识别和量化神经元培养和完整动物中突触蛋白质组的变化的可行性。

DESCRIPTION (provided by applicant): Synapses are dynamic, with respect to their magnitude of synaptic transmission, their morphologic structure and their protein components. Dynamic changes in the synaptic proteome, mediated by changes in protein synthesis, degradation, distribution and post- translational modifications affect synaptic function and the capacity for synaptic plasticity. Despite the appreciation of the biological importance of synaptic protein dynamics, methods to enrich for and identify the dynamic synaptic proteome are not yet an integral part of neuroscience research. Studies conducted largely in prokaryotes and non-neuronal cells have demonstrated the promise of using the incorporation of non- canonical amino acids into newly synthesized proteins followed by bio-orthogonal click chemistry to tag the non-canonical amino acid labeled protein with probes, such as biotin, that can be used for purification and identification of newly synthesized proteins. We propose experiments to optimize the use of non-canonical amino acid incorporation and bio-orthogonal click chemistry in neuronal cell cultures, and in the visual system of intact rats and Xenopus tadpoles to demonstrate the feasibility of metabolic labeling to identify and quantify dynamic components of the synaptic proteome. These experiments are meant to represent proof of principal for comparing synthesis of synaptic proteins between different conditions and in different experimental systems. PUBLIC HEALTH RELEVANCE: Newly synthesized synaptic proteins regulate synaptic function and plasticity. We propose to improve feasibility of using metabolic labeling with non-canonical amino acids to identify and quantify changes in the synaptic proteome in neuronal cultures and intact animals.