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）：自从电子显微镜的早期发现以来，毛细胞突触色带的结构和功能一直是神秘的。假定它在确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起着关键作用，但是尚不清楚它是否具有其他功能。自发现以来的半个世纪中，我们对其分子解剖结构或功能的了解相对较少。尽管在色带中存在许多已知在常规突触中很重要的蛋白质，但我们对结构本身的主要成分知之甚少。基于质谱的蛋白质组学方法最近开发了用于蛋白质鉴定的蛋白质组学方法，结合了相对完整的小鼠蛋白质组，可以在很小的样品中鉴定数百种蛋白质。因此，在纯化的突触色带样品中重新鉴定蛋白质鉴定应产生大部分（如果不是全部的蛋白质），并与突触色带相关。这项工作很简单，过程已经到位，小鼠蛋白质组高度发达，并且质量规格数据的蛋白质组学分析是常规的。具体目的是（1）鉴定纯化的突触色带复合物中的蛋白质成分以及色带的电子致密核心。 （2）免疫化学在小鼠内耳的突触带和从牛视网膜纯净的丝带配合物中验证候选物的存在。显然，理解像色带一样复杂的结构功能的先决条件是定义蛋白质组学水平的色带的结构。对于许多确定的蛋白质，基于对其他系统中蛋白质的分析，功能将是隐式的。其他人可能需要一些思考或实验来确定他们的工作。似乎毫无疑问，这种直接的识别突触色带中的蛋白质应该为丝带所做的事情产生许多假设，这可能会使研究人员在未来的一段时间内忙碌。鉴定突触色带的分子成分将是了解其在传入突触中特定功能的基本步骤。公共卫生相关性：突触色带在半个世纪前的发现以来，突触丝带在毛细胞突触传播中的作用一直是神秘的。蛋白质组学的最新进展将允许鉴定这种异常的亚细胞结构的分子结构，这对于正常听力至关重要。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面板应该对其功能进行特定的，可检验的假设，该假设可能会为一系列听觉疾病提供见解，包括耳鸣和噪声引起的听力损失。