Proteomic Analysis of Synaptic Vesicle Pools

突触小泡池的蛋白质组学分析

• 批准号: 8690166
• 负责人: ROBERT H EDWARDS
• 金额: $ 19.75万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8690166
• 关键词: Behavior; Biochemical; Cell membrane; Chemicals; Chemistry; Cytolysis; Cytoskeleton; Development; Docking; Electron Microscopy; Exhibits; Foundations; Future; Individual; Label; Magnetism; Mammals; Mass Spectrum Analysis; Membrane Proteins; Molecular; Morphology; Neurons; Organelles; Pattern; Population; Procedures; Process; Property; Proteins; Proteomics; Protocols documentation; Recording of previous events; Recovery; Recycling; Relative (related person); Reporter; Rest; Site; Stable Isotope Labeling; Surface; Synapses; Synaptic Transmission; Synaptic Vesicles; Synaptosomes; System; Vesicle; Work; base; nanoparticle; neurotransmitter release; neurotransmitter uptake; optical imaging; public health relevance; research study; response; theories

DESCRIPTION (provided by applicant): Classical studies in the field of synaptic transmission have assumed that neurotransmitter release occurs from a biochemically homogeneous population of synaptic vesicles, but considerable work from many experimental systems has shown that synaptic vesicles belong to pools that differ in their response to stimulation. These observations have given rise to two competing hypotheses, one that the pools are biochemically the same, with differences in behavior strictly stochastic, or extrinsic, reflecting differential association with the cytoskeleton or prior history rather than any intrinsic differences in composition. Alternatively, differences in molecular composition underlie the behavior of different synaptic vesicle pools, and recent work has suggested that the pools may retain their identity after recycling. Although controversial, we have recently shown that different synaptic vesicle proteins respond differently to stimulation, providing some of the first evidence that synaptic vesicle pools differ in composition. However, these experiments involved optical imaging of individual reporter constructs, and understanding how membrane protein composition determines the properties of synaptic vesicles requires a more systematic approach. We will thus label specific synaptic vesicle pools with magnetic nanoparticles strictly on the basis of their response to activity, and determine their composition by quantitative proteomic analysis: Aim 1: Optimize synaptic vesicle recovery from highly purified synaptoneurosomes. Standard procedures fail to recover synaptic vesicles associated with the plasma membrane, so we will optimize synaptic vesicle recovery from synaptoneurosomes using a combination of physical and chemical approaches. Aim 2: Optimize isolation of synaptic vesicles labeled with magnetic nanoparticles during stimulation. We will synthesize small magnetic nanoparticles, and optimize the labeling of different synaptic vesicle pools by different patterns of stimulation. Aim 3: Determine the composition of recycling and resting synaptic vesicle pools by quantitative proteomics using isobaric tag for relative and absolute quantitation (iTRAQ) or stable isotope labeling in mammals (SILAM). Identifying the molecular composition of different synaptic vesicle pools will provide a foundation for future work to explore the functin of the identified components in transmitter release, synapse development and plasticity.

描述（由申请人提供）：突触传递领域的经典研究假设神经递质释放发生在生化同质的突触小泡群体中，但许多实验系统的大量工作表明，突触小泡属于对刺激反应不同的池。这些观察结果产生了两种相互竞争的假设，一种假设是池在生化上是相同的，但行为上的差异是严格随机的或外在的，反映了与细胞骨架或先前历史的差异关联，而不是成分上的任何内在差异。或者，分子组成的差异是不同突触小泡池行为的基础，最近的研究表明，这些池在回收后可能保留其特性。尽管存在争议，但我们最近表明，不同的突触小泡蛋白对刺激的反应不同，这提供了突触小泡池的组成不同的一些初步证据。然而，这些实验涉及单个报告基因构建体的光学成像，并且了解膜蛋白组成如何决定突触小泡的特性需要更系统的方法。因此，我们将严格根据其对活性的反应，用磁性纳米粒子标记特定的突触小泡池，并通过定量蛋白质组分析确定其组成： 目标 1：优化从高度纯化的突触神经小体中回收突触小泡。标准程序无法回收与质膜相关的突触小泡，因此我们将使用物理和化学方法相结合来优化从突触神经小体中回收突触小泡。目标 2：在刺激过程中优化磁性纳米颗粒标记的突触小泡的分离。我们将合成小型磁性纳米粒子，并通过不同的刺激模式优化不同突触囊泡池的标记。目标 3：使用同量异位标签进行相对和绝对定量 (iTRAQ) 或哺乳动物稳定同位素标记 (SILAM)，通过定量蛋白质组学确定回收和静息突触囊泡池的组成。识别不同突触小泡池的分子组成将为未来探索已识别成分在递质释放、突触发育和可塑性中的功能奠定基础。