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器官。每个毛细胞都有一束以肌动蛋白为基础的立体纤毛，排列高度增加；细胞的每根立体纤毛延伸一根丝状的“尖端连接”到下一根更高的立体纤毛。束的运动使尖端环节收紧；它们反过来打开强制门控离子通道，打开离子通道使细胞去极化。因此，耳尖连接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，排列在反平行的异四聚体细丝中。虽然经典钙粘蛋白的同源结合是已知的，但这些尖端连接的钙粘蛋白缺乏介导这种结合的关键氨基酸。此外，这两种蛋白的突变都会导致以先天性耳聋和进行性失明为特征的Usher综合征，但目前尚不清楚这些突变是如何导致听力损失的。我们将研究这些钙粘蛋白之间的结合，通过单独解决远端晶体结构，然后作为异四聚体复合物。我们已经解决了钙粘蛋白23 n端的四种结构：野生型和突变型蛋白质，高和低Ca2+浓度。我们将扩展到原钙粘蛋白15n端，以了解Ca2+和突变如何影响结合和尖端连接的完整性。有了晶体结构在手，我们将使用定向分子动力学计算来了解这些钙粘蛋白如何在高张力下展开，以及Ca2+浓度和耳聋引起的突变如何影响展开力。最初的研究表明，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，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质结合界面，通过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之间的分子结构将通过诱变实验进行测试，以确定哪些氨基酸对体外结合至关重要，哪些氨基酸需要通过限制自由末端来防止尖端连接的再生。

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.