Molecular Basis of Inherited Deafness

遗传性耳聋的分子基础

• 批准号: 8227964
• 负责人: DAVID P COREY
• 金额: $ 35.91万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-07-01 至 2016-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8227964
• 关键词: Actins; Affect; Amino Acids; Auditory; Binding; Biochemical; Biological Assay; Blindness; Cadherins; Cells; Complex; Crystallization; Distal; Excision; Filament; Gated Ion Channel; Genes; Goals; Hair Cells; Hand; Head Movements; Heart; Height; Human; In Vitro; Inborn Genetic Diseases; Inherited; Labyrinth; Link; Mediating; Methods; Molecular; Molecular Structure; Movement; Mus; Mutagenesis; Mutate; Mutation; N-terminal; Natural regeneration; Noise; Proteins; Receptor Cell; Recombinants; Sensory; Signal Transduction; Stereocilium; Structure; System; Testing; Usher Syndrome; Work; X-Ray Crystallography; base; congenital deafness; deafness; design; hair cell regeneration; hearing impairment; human CDH23 protein; insight; molecular dynamics; mutant; prevent; public health relevance; relating to nervous system; research study; response; sound

DESCRIPTION (provided by applicant): This work is designed to understand how proteins encoded by two deafness genes-cadherin 23 and protocadherin 15-assemble to form the mechanosensory apparatus of hair cells in the auditory and vestibular systems. Each hair cell has a bundle of actin-based stereocilia arranged with increasing heights; each stereocilium of a cell extends a filamentous 'tip link' to the next taller stereocilium. Movement of the bundle tightens tip links; they in turn pull open force-gated ion channels that open to depolarize the cell. Thus tip links are a key component at the heart of inner ear function-to turn sound and head movement into neural signals. Recent evidence indicates that each tip link is composed of cadherin 23 and protocadherin 15 arranged in an antiparallel hetero-tetrameric filament. While the homomeric binding of classical cadherins is understood, these tip-link cadherins lack the key amino acids that mediate such binding. Moreover, mutations in either protein cause Usher Syndrome, characterized by congenital deafness and progressive blindness, but it is not known how these mutations cause hearing loss. We will investigate the binding between these cadherins, by solving the crystal structures of the distal ends individually and then as a heterotetrameric complex. We have already solved four structures for the cadherin 23 N-terminus: for wild-type and mutant forms of the proteins, and in high and low Ca2+ concentration. We will extend this to the protocadherin 15 N terminus, to understand how both Ca2+ and mutations affect binding and tip-link integrity. With crystal structures in hand, we will use steered molecular dynamics calculations to understand how these cadherins unfold in response to high tension, and how Ca2+ concentration and deafness-causing mutations affect the unfolding force. Initial work shows that removing Ca2+ or mutating Ca2+-binding residues allows cadherins to unfold at lower force, making them more susceptible to loud noise. We will also use molecular dynamics to explore the predicted heteromeric binding interface by asking what forces are needed to pull apart the tip link and how Ca2+ maintains that bond. The molecular structure of the bond between cadherin 23 and protocadherin 15 suggested by these studies will be tested by mutagenesis experiments, to see which amino acids are critical for binding in vitro and which are required to prevent regeneration of tip links by capping the free ends. PUBLIC HEALTH RELEVANCE: Usher's Syndrome, a devastating inherited disorder characterized by congenital deafness and progressive blindness, is caused by mutations in genes encoding the proteins cadherin 23 and protocadherin 15. This project is directed at understanding what these cadherins do in the inner ear; specifically, which forms are part of the sensory apparatus of cochlear receptor cells, and how they assemble to create the apparatus. The results will help us design strategies for treating inherited deafness.

描述（由申请人提供）：这项工作旨在了解由两个耳聋基因钙粘蛋白 23 和原钙粘蛋白 15 编码的蛋白质如何组装形成听觉和前庭系统中毛细胞的机械感觉装置。 每个毛细胞都有一束基于肌动蛋白的静纤毛，其高度逐渐增加；细胞的每个静纤毛都将丝状“尖端连接”延伸到下一个更高的静纤毛。束的运动收紧尖端链接；它们反过来拉开力门控离子通道，使细胞去极化。 因此，尖端连杆是内耳功能核心的关键组成部分——将声音和头部运动转化为神经信号。 最近的证据表明，每个尖端连接由钙粘蛋白 23 和原钙粘蛋白 15 组成，排列成反向平行的异源四聚体丝。 虽然经典钙粘蛋白的同聚结合已被了解，但这些尖端连接钙粘蛋白缺乏介导这种结合的关键氨基酸。 此外，任一蛋白质的突变都会导致亚瑟综合症，其特征是先天性耳聋和进行性失明，但尚不清楚这些突变如何导致听力损失。 我们将通过单独解析远端的晶体结构，然后作为异四聚体复合物来研究这些钙粘蛋白之间的结合。 我们已经解决了钙粘蛋白 23 N 末端的四种结构：蛋白质的野生型和突变型，以及高和低 Ca2+ 浓度。 我们将其扩展到原钙粘蛋白 15 N 末端，以了解 Ca2+ 和突变如何影响结合和尖端连接完整性。 掌握晶体结构后，我们将使用引导分子动力学计算来了解这些钙粘蛋白如何响应高张力而展开，以及 Ca2+ 浓度和导致耳聋的突变如何影响展开力。 初步研究表明，去除 Ca2+ 或突变 Ca2+ 结合残基可以使钙粘蛋白以较低的力展开，从而使它们更容易受到噪音的影响。 我们还将利用分子动力学来探索预测的异聚结合界面，询问需要什么力来拉开尖端连接以及 Ca2+ 如何维持该键。 这些研究提出的钙粘蛋白 23 和原钙粘蛋白 15 之间键的分子结构将通过诱变实验进行测试，以了解哪些氨基酸对于体外结合至关重要，以及哪些氨基酸是通过封盖自由端来防止尖端连接再生所必需的。 公共健康相关性：亚瑟氏综合症是一种破坏性遗传性疾病，其特征是先天性耳聋和进行性失明，是由编码钙粘蛋白 23 和原钙粘蛋白 15 的基因突变引起的。该项目旨在了解这些钙粘蛋白在内耳中的作用；具体来说，哪些形式是耳蜗感受器细胞感觉装置的一部分，以及它们如何组装以创建该装置。 研究结果将帮助我们设计治疗遗传性耳聋的策略。