Towards a Dendritic Proteome

迈向树突状蛋白质组

• 批准号: 7229840
• 负责人: ERIN M SCHUMAN
• 金额: $ 19.34万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 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/7229840
• 关键词: Affinity; Agonist; Alkynes; Amino Acids; Area; Axon; Biochemical; Bioinformatics; Cells; Chromosome Pairing; Condition; Coupling; Data Set; Dendrites; Destinations; Development; Electric Stimulation; Environment; Generations; Genetic Transcription; Genetic Translation; Green Fluorescent Proteins; Growth Factor; Hippocampus (Brain); Histocompatibility Testing; Hot Spot; Image; In Situ; Individual; Label; Laboratories; Ligation; Localized; Logic; Maintenance; Messenger RNA; Methods; Modification; Neurons; Neurotransmitters; Numbers; Phase; Polymerase Chain Reaction; Positioning Attribute; Preparation; Procedures; Process; Protein Binding; Protein Biosynthesis; Proteins; Proteome; Proteomics; Relative (related person); Reporter; Ribosomes; Site; Specificity; Synapses; Synaptic Transmission; Synaptic plasticity; Techniques; Technology; Time; Translating; Translations; Vertebral column; addiction; base; environmental change; interest; neuronal cell body; neurotransmitter release; polarized cell; protein expression; research study; response; tandem mass spectrometry

DESCRIPTION (provided by applicant): In neurons, there is increasing evidence that local dendritic protein synthesis is used to allow individual synapses to respond dynamically to the environmental changes that accompany the establishment, maintenance and plasticity of synaptic connections. Furthermore, it is now well established that the essential components of the translational machinery as well as mRNAs are present at or near synapses and that dendrites can synthesize new proteins. Despite this, only a limited number of locally translated proteins have been identified so far, mainly through candidate-based approaches inspired by a particular laboratory's interest in protein "x" or "y" ¿r by mRNA generation from single neuritic process with subsequent PCR experiments. However, we believe that a focus on translated proteins is essential for an accurate picture of how the synapse and the dendrite can mount a response to local changes in the environment. Current available methods to evaluate changes in the protein composition of a cell or of a cellular compartment rely on the comparison of two proteomes with one another. These differential approaches are successful only when proteins are present in sufficient quantity to be detected and when there are large differences in protein expression levels between the compared proteomes. Here we describe the development of a new high-throughput proteomic profiling technique to isolate and identify the translation products in neuronal dendrites and to determine the quantitative translational capability of dendrites under both basal and stimulated conditions. Specifically, we propose a procedure that uses artificial amino acids for the labeling of newly synthesized dendritic proteins in situ, thereby circumventing problems of traditional approaches. The obtained proteomic datasets will help us to understand the logic of local translation. This technology can be easily applied to many proteomic profiling questions, regardless of tissue type or cellular context. The changes in dendritic proteins that underly the synaptic modifications associated with plasticity, including addiction, can be elucidated using this technique.

描述（由申请人提供）：在神经元中，越来越多的证据表明，局部树突蛋白合成用于允许单个突触动态响应伴随突触连接的建立、维持和可塑性的环境变化。此外，现在已经确定翻译机制的基本成分以及mrna存在于突触或突触附近，并且树突可以合成新的蛋白质。尽管如此，到目前为止，只有有限数量的局部翻译蛋白被鉴定出来，主要是通过基于候选蛋白的方法，这些方法的灵感来自于一个特定实验室对蛋白质“x”或“y”¿r的兴趣，通过随后的PCR实验从单个神经性过程中产生mRNA。然而，我们相信，对翻译蛋白的关注对于准确了解突触和树突如何对环境的局部变化作出反应是必不可少的。目前可用的方法来评估在一个细胞或细胞室的蛋白质组成的变化依赖于两个蛋白质组相互比较。这些差异方法只有在蛋白质存在足够数量以检测到并且在比较的蛋白质组之间蛋白质表达水平存在很大差异时才能成功。在这里，我们描述了一种新的高通量蛋白质组学分析技术的发展，以分离和鉴定神经元树突中的翻译产物，并确定树突在基础和刺激条件下的定量翻译能力。具体来说，我们提出了一种使用人工氨基酸原位标记新合成树突状蛋白的方法，从而避免了传统方法的问题。获得的蛋白质组学数据集将有助于我们理解局部翻译的逻辑。这项技术可以很容易地应用于许多蛋白质组学分析问题，而不考虑组织类型或细胞背景。树突蛋白的变化是与可塑性相关的突触修饰的基础，包括成瘾，可以用这种技术来阐明。