Molecular Basis of 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。 每个毛细胞有一束肌动蛋白为基础的静纤毛排列与增加的高度;每个静纤毛的细胞延伸丝状的“尖端链接”到下一个更高的静纤毛。纤维束的运动会使尖端连接收紧，它们反过来又会打开力门控离子通道，从而打开细胞。 因此，耳尖链接是内耳功能的核心关键组成部分-将声音和头部运动转化为神经信号。 最近的证据表明，每个尖端链接是由钙粘蛋白23和原钙粘蛋白15排列在一个反平行的异源四聚体丝。 虽然经典钙粘蛋白的同聚体结合被理解，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，任何一种蛋白质的突变都会导致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。 我们将研究这些钙粘蛋白之间的结合，通过单独解决的远端的晶体结构，然后作为一个异源四聚体复合物。 我们已经解决了四个结构的钙粘蛋白23 N-末端：野生型和突变形式的蛋白质，并在高和低的Ca 2+浓度。 我们将把它扩展到原钙粘蛋白15 N末端，以了解Ca 2+和突变如何影响结合和尖端连接完整性。 有了晶体结构，我们将使用操纵分子动力学计算来了解这些钙粘蛋白如何响应高张力展开，以及Ca 2+浓度和导致突变的突变如何影响展开力。 最初的工作表明，去除Ca 2+或突变Ca 2+结合残基可以使钙粘蛋白在较低的力下展开，使它们更容易受到噪音的影响。 我们还将使用分子动力学来探索预测的异聚体结合界面，询问需要什么力量来拉开尖端链接以及Ca 2+如何保持这种键。 这些研究所提出的钙粘蛋白23和原钙粘蛋白15之间的键的分子结构将通过诱变实验进行测试，以观察哪些氨基酸对于体外结合是关键的，以及哪些氨基酸是通过加帽自由端来防止尖端连接再生所需的。

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.