Structural and Functional Characterization of Otoferlin

描述(由申请人提供)：本项目的目的是确定听力相关蛋白Otoferlin的功能和结构特性。这种蛋白质被认为直接调节膜融合和由此产生的耳蜗内毛细胞神经递质的释放。耳铁蛋白的突变会导致出生时严重的听力损失。然而，耳铁蛋白在听力中的确切作用还不是很清楚。在这个奖项下追求的以下具体目标旨在提高我们对听觉通路中的Otoferlin的理解。如果Otoferlin确实直接参与胞吐作用，它应该具有某些特有的功能特性，使其能够参与膜融合事件。在第一个目标中，将首次使用体外融合实验直接测试耳铁蛋白与SNARE蛋白结合和促进膜融合的假想能力。这将确立耳铁蛋白在胞吐过程中的功能作用。还将探讨奥托费林的钙结合特性，以试图确定该蛋白质是否为钙传感器。第二个目的将利用X射线结晶学方法阐明蛋白质的结构，以便为观察到的功能性质提供结构基础。还将检查该蛋白质的几种病理突变形式，以确定病理的分子基础。第三，将阐明奥托费林与脂膜的关系。操纵脂双分子层是参与膜融合和胞吐的蛋白质的共同特征。然而，目前尚不清楚奥托费林是否真的能结合并改变膜的形态。这一目标将检验奥托费林能够以钙依赖的方式穿透和诱导脂质双层弯曲的假说，作为加速膜融合的机制步骤。 与公共健康相关：这项建议试图了解耳铁蛋白在听力中所起的作用，以及为什么这种蛋白的突变会导致耳聋。将进行功能和结构研究，以更好地了解这种耳蜗特异性蛋白质如何能够控制神经递质的释放。

DESCRIPTION (provided by applicant): The aim of this project is to determine the functional and structural properties of the hearing associated protein otoferlin. This protein is believed to directly regulate membrane fusion and the resultant neurotransmitter release from inner hair cells in the cochlea. Mutations in otoferlin result in profound hearing loss at birth. However, the exact role of otoferlin in hearing is not well understood. The following specific aims pursued under this award are intended to improve our understanding of otoferlin in the auditory pathway. If otoferlin is indeed directly involved in exocytosis, it should posses certain characteristic functional properties which allow it to partake in membrane fusion events. In the first aim, the hypothesized ability of otoferlin to bind SNARE proteins and accelerate membrane fusion will be directly tested for the first time using an in vitro fusion assay. This will establish a functional role for otoferlin in the exocytosis pathway. The calcium binding properties of otoferlin will also be probed in an attempt to determine whether the protein is a calcium sensor. The second aim will elucidate the structure of the protein using X-ray crystallographic methods so as to provide a structural basis for the observed functional properties. Several pathological mutant forms of the protein will also be examined so as to determine the molecular basis for the pathology. Third, the relationship between otoferlin and lipid membranes will be elucidated. Manipulation of lipid bilayers is a common characteristic of proteins involved in membrane fusion and exocytosis. Yet it is currently unknown whether otoferlin can indeed bind to and alter membrane morphology. This aim will test the hypothesis that otoferlin can penetrate and induce curvature in lipid bilayers in a calcium dependent manner as a mechanistic step in accelerating membrane fusion. PUBLIC HEALTH RELEVANCE: This proposal seeks to understand the role that the protein otoferlin plays in hearing and why mutations in this protein result in deafness. Both functional and structural studies will be undertaken to provide a better understanding as to how this cochlea-specific protein is able to control neurotransmitter release.