Molecular Basis of Inherited Deafness

遗传性耳聋的分子基础

基本信息

批准号：
8610278
负责人：
DAVID P COREY
金额：
$ 35.98万
依托单位：
HARVARD MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
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

项目摘要

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.

描述（由申请人提供）：这项工作旨在了解如何由两个耳聋基因 - 钙粘蛋白23和原粘蛋白15组装编码，以形成听觉和前庭系统中毛细胞的机械感应设备。每个毛细胞都有一束基于肌动蛋白的立体胶质，并以增加身高增加。一个细胞的每个立体胶体都将丝状“尖端链接”延伸到下一个更高的立体胶质。束的运动收紧尖端链接；他们反过来拉动开放力的离子通道，以使细胞去极化。因此，尖端链接是内耳功能核心的关键组件，将声音和头部运动转向神经信号。最近的证据表明，每个尖端链路由钙粘蛋白23和原钙粘蛋白15组成，该蛋白质在反平行的杂型四聚体细丝中排列。尽管可以理解经典钙粘蛋白的同质结合，但这些尖端连接蛋白蛋白缺乏介导这种结合的关键氨基酸。此外，这两种蛋白质的突变引起了usher综合征，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。我们将通过单独求解远端末端的晶体结构，然后作为异射术配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末端的四个结构：用于蛋白质的野生型和突变形式，以及高和低Ca2+浓度。我们将将其扩展到原始钙粘蛋白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。借助晶体结构，我们将使用转向的分子动力学计算来了解这些核蛋白如何响应高张力，以及Ca2+浓度和聋哑突变如何影响展开力。最初的工作表明，去除Ca2+或突变Ca2+结合残基会使钙粘蛋白在较低力下展开，从而使它们更容易受到大声噪声的影响。我们还将使用分子动力学来探索预测的杂体结合界面，询问需要哪些力才能拉开尖端链接以及CA2+如何保持该键。这些研究提出的钙粘蛋白23和原粘蛋白15之间的键的分子结构将通过诱变实验进行测试，以查看哪些氨基酸对于结合体外至关重要，哪些氨基酸是通过限制自由末端来防止尖端链接再生所必需的。