Structural and Functional Characterization of Otoferlin

R&R其他项目信息第6项：汇总/摘要 该项目的目的是确定功能和结构属性 听力相关蛋白Otoferlin。这种蛋白质被认为直接调节细胞膜。 融合和由此产生的神经递质从耳蜗内毛细胞释放。突变 在出生时会导致严重的听力损失。然而，耳铁蛋白在听力中的确切作用 还没有被很好地理解。根据本奖项追求的下列具体目标旨在 提高我们对听觉通路中的耳铁蛋白的理解。 如果Otoferlin确实直接参与胞吐作用，它应该具有一定的特征 允许其参与膜融合事件的功能特性。第一个目标是， 推测耳铁蛋白结合SNARE蛋白和促进膜融合的能力将是 首次使用体外融合试验进行了直接检测。这将建立一个职能角色 对于胞吐作用途径中的耳铁蛋白。奥托费林的钙结合特性也将是 试图确定这种蛋白质是否是钙传感器。 第二个目标将利用X射线结晶学来阐明蛋白质的结构 方法，以便为观察到的功能特性提供结构基础。几个 还将检查蛋白质的病理突变形式，以确定分子 病理基础。 第三，将阐明奥托费林与脂膜的关系。 膜相关蛋白质的共同特征是操纵脂双分子层 融合和胞吐。然而，目前尚不清楚奥托费林是否真的可以结合和改变 膜形态。这一目标将检验奥托费林可以穿透并诱导 钙依赖的脂质双层的曲率作为加速的机械性步骤 膜融合。

Item 6 of R&R Other Project Information: Summary/Abstract 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.