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，局部树突蛋白合成被用来允许个体突触动态地响应伴随突触连接的建立、维持和可塑性的环境变化。确定翻译机制的基本成分以及 mRNA 存在于突触处或突触附近，并且树突可以合成新蛋白质，尽管如此，迄今为止仅鉴定了有限数量的局部翻译蛋白质，主要通过基于候选的方法，灵感来自于特定实验室对蛋白质“x”或“y”的兴趣 ¿然而，我们认为，重点关注翻译蛋白对于准确了解突触和树突如何对环境中的局部变化做出反应至关重要。评估细胞或细胞区室的蛋白质组成的变化依赖于两种蛋白质组的相互比较，只有当蛋白质存在足够的量以供检测并且蛋白质存在较大差异时，这些差异方法才能成功。比较组之间的表达水平在这里，我们描述了一种新的高通量蛋白质组分析技术的开发，用于分离和鉴定神经树突中的翻译产物，并确定树突在基础和刺激条件下的定量翻译能力。人工氨基酸用于原位标记新合成的树突状蛋白，从而规避传统方法的问题，所获得的蛋白质组数据集将帮助我们轻松理解本地翻译的逻辑。应用于许多蛋白质组分析问题，无论组织类型或细胞环境如何，都可以使用该技术来阐明与可塑性（包括成瘾）相关的突触修饰的树突蛋白的变化。