Structural and Functional Characterization of Otoferlin

R&R其他项目信息第6项：摘要/摘要 本项目的目的是确定的功能和结构特性的 听觉相关蛋白耳铁蛋白。这种蛋白质被认为是直接调节膜 融合和由此产生的神经递质从耳蜗内毛细胞释放。突变 导致出生时严重听力损失。然而，otoferlin在听力中的确切作用 并没有得到很好的理解。本奖项追求的以下具体目标旨在 提高我们对听觉通路中otoferlin的理解。 如果otoferlin确实直接参与胞吐作用，它应该具有某些特征， 使其参与膜融合事件的功能特性。在第一个目标中， 假设otoferlin结合SNARE蛋白和加速膜融合能力将 首次使用体外融合测定直接测试。这将建立一个职能作用 在胞吐途径中的otoferlin。otoferlin的钙结合特性也将被证实。 试图确定蛋白质是否是钙传感器。 第二个目标是用X射线晶体学方法阐明蛋白质的结构 方法，以便为观察到的功能特性提供结构基础。几 还将检查蛋白质的病理突变形式，以确定其分子结构。 病理学的基础。 第三，otoferlin和脂质膜之间的关系将被阐明。 操纵脂质双层是膜蛋白的共同特征 融合和胞吐作用。然而，目前尚不清楚otoferlin是否真的可以结合并改变 膜形态学这一目的将检验otoferlin可以渗透并诱导 脂质双层中的曲率以钙依赖的方式作为加速的机械步骤 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.