Proteomic analysis of synaptic ribbons in the inner ear

内耳突触带的蛋白质组学分析

• 批准号: 7511325
• 负责人: WILLIAM F SEWELL
• 金额: $ 22.65万
• 依托单位: MASSACHUSETTS EYE AND EAR INFIRMARY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7511325
• 关键词: Acoustic Nerve; Amaze; Anatomy; Auditory; Bos taurus; Cattle; Cells; Characteristics; Chemicals; Chimeric Proteins; Chromosome Pairing; Complex; Coupled; Data; Development; Disease; Electron Microscopy; Electrons; Enzymes; Hair Cells; Hearing; Labyrinth; Learning; Molecular; Molecular Structure; Mus; Nerve Fibers; Noise Induced Tinnitus; Noise-Induced Hearing Loss; Play; Procedures; Protein Analysis; Protein Isoforms; Proteins; Proteome; Proteomics; Public Health; Range; Research Personnel; Retina; Retinal Photoreceptors; Role; Sampling; Shapes; Structure; Subcellular structure; Surface; Synapses; System; Thinking; Time; Two-Dimensional Gel Electrophoresis; Work; base; day; immunoreactivity; insight; interest; neurotransmission; presynaptic; protein purification; ribbon synapse; transmission process

DESCRIPTION (provided by applicant): Both the structure and the function of the hair cell's synaptic ribbon have been enigmatic since its discovery in the early days of electron microscopy. It is assumed that it plays a key role in determining the amazing temporal and dynamic characteristics of afferent neurotransmission between the hair cell and the auditory nerve fiber, but whether it has other functions is not known. In the half-century since its discovery, we have learned relatively little about its molecular anatomy or its function. Though many proteins known to be important in conventional synapses are present at the ribbon, we know little about the dominant components of the structure itself. The recent development of mass-spectrometric-based proteomics approaches for protein identification, coupled to a relatively complete proteome for the mouse, allows identification of hundreds of proteins in very small samples. Thus revisiting protein identification in purified samples of synaptic ribbon should yield most if not all of the proteins in and associated with the synaptic ribbon. The work is straightforward, the procedures are in place, the mouse proteome is highly developed, and the proteomic analysis of mass spec data is routine. The specific aims are to (1) Identify protein components in purified synaptic ribbon complexes, and in the electron dense core of the ribbon. (2) Immunochemically verify presence of candidates in synaptic ribbons of the mouse inner ear and in ribbon complexes purified from bovine retina. It seems evident that a prerequisite to understanding function of a structure as complex as the ribbon is to define the structure of the ribbon at the proteomics level. For many proteins identified, function will be implicit, based on analysis of the protein in other systems. Others may need some thought or experimentation to determine what they do. There seems no question that this straightforward quest to identify proteins in the synaptic ribbon should generate many hypotheses for what the ribbon is doing that could keep investigators busy for some time to come. Identification of the molecular components of the synaptic ribbon will be a fundamental step towards understanding its specific function at the afferent synapse. PUBLIC HEALTH RELEVANCE: The synaptic ribbon's role in transmission at the hair cell synapse has been enigmatic since its discovery over a half century ago. Recent advances in proteomics will allow identification of the molecular structure of this unusual subcellular structure that is essential for normal hearing. Because most proteins have specific molecular functions, knowing the panel of players in this structure should engender specific, testable hypotheses as to its function that may provide insights into a range of auditory disorders, including tinnitus and noise-induced hearing loss.

描述（由申请人提供）：自从电子显微镜早期发现毛细胞突触带的结构和功能以来，它的结构和功能一直是个谜。据认为，它在决定毛细胞和听觉神经纤维之间传入神经传递的惊人时间和动态特征方面发挥着关键作用，但它是否具有其他功能尚不清楚。自发现以来的半个世纪里，我们对其分子解剖学或其功能了解相对较少。尽管许多已知在传统突触中重要的蛋白质都存在于带上，但我们对该结构本身的主要成分知之甚少。最近开发的基于质谱的蛋白质组学方法用于蛋白质鉴定，与相对完整的小鼠蛋白质组相结合，可以在非常小的样品中鉴定数百种蛋白质。因此，重新审视突触带纯化样品中的蛋白质鉴定应该会产生突触带中的大部分（如果不是全部）蛋白质以及与突触带相关的蛋白质。这项工作很简单，程序到位，小鼠蛋白质组高度发达，质谱数据的蛋白质组分析是常规的。具体目标是 (1) 鉴定纯化的突触带复合物以及带的电子致密核心中的蛋白质成分。 (2) 通过免疫化学方法验证小鼠内耳突触带和从牛视网膜纯化的带状复合物中是否存在候选物。显然，理解像带一样复杂的结构的功能的先决条件是在蛋白质组学水平上定义带的结构。对于许多已识别的蛋白质，基于对其他系统中蛋白质的分析，功能将是隐含的。其他人可能需要一些思考或实验来确定他们要做什么。毫无疑问，这种识别突触带中蛋白质的直接探索应该会产生许多关于突触带正在做什么的假设，这可能会让研究人员在未来一段时间内忙碌起来。突触带分子成分的识别将是理解其在传入突触的特定功能的基本步骤。公共健康相关性：自半个多世纪前发现以来，突触带在毛细胞突触传递中的作用一直是个谜。蛋白质组学的最新进展将有助于鉴定这种对正常听力至关重要的不寻常亚细胞结构的分子结构。由于大多数蛋白质具有特定的分子功能，因此了解该结构中的参与者应该可以对其功能产生特定的、可测试的假设，从而可以深入了解一系列听觉障碍，包括耳鸣和噪音引起的听力损失。