Biochemical consequences of Deafness-causing actin mutations

描述(申请人提供)：毛细胞位于内耳，是听力过程的重要组成部分。肌动蛋白细胞骨架在毛细胞功能中起主要作用，它通过位于立体纤毛的肌动蛋白细丝束和位于角质板的肌动蛋白网络。已发现6个非肌肉肌动蛋白(DFNA 20/26)突变导致常染色体显性遗传性非综合征性听力损失。然而，这些突变导致耳聋的分子基础尚不清楚，很大程度上是因为无法获得足够的突变肌动蛋白，以在生化水平上评估突变对肌动蛋白功能的影响。为了解决这个问题并了解这些突变的生化后果，我们将每个突变克隆到酵母肌动蛋白中，酵母肌动蛋白与人类伽玛非肌肉肌动蛋白有91%的同源性，我们可以纯化足够数量的突变肌动蛋白用于生化分析。这些突变会在酵母中引起等位基因特异性的影响，但只有一个突变会在体外影响纯肌动蛋白的聚合。这一结果表明，突变可能会干扰肌动蛋白由不同的肌动蛋白细丝调节蛋白控制的能力。我们将使用成熟的肌动蛋白聚合的生化和荧光显微镜分析来评估这六种导致耳聋的突变对肌动蛋白与肌动蛋白结合蛋白相互作用的影响，肌动蛋白结合蛋白可能调节毛细胞中的肌动蛋白细丝功能。具体地说，我们将继续使用酵母肌动蛋白系统来检测突变对cofilin、肌动蛋白细丝切割蛋白、Arp2/3复合体和福尔马林的影响，这些蛋白启动肌动蛋白聚合和控制肌动蛋白细丝长度。我们将研究突变对肌动蛋白与三种与耳聋相关的肌动蛋白细丝束蛋白以及ESpin、1-catenin和Eps8相互作用能力的影响。我们还将使用质谱学结合氢-氚交换来直接确定突变对肌动蛋白细丝构象和灵活性的影响。最后，我们将建立杆状病毒表达系统，以产生包含耳聋突变的哺乳动物非肌肉3-异肌动蛋白，以便评估在毛细胞中实际发生的肌动蛋白突变的影响。相关性：在美国，耳聋是一个主要的健康问题。这项拟议的工作将深入了解这六种肌动蛋白突变导致耳聋的生化基础。这将导致对纯3-非肌肉等肌动蛋白的性质进行严格的系统检查。此外，关于这些突变如何影响肌动蛋白与肌动蛋白调节蛋白相互作用的信息将为肌动蛋白在正常耳朵中如何发挥作用和在生化水平上受到调控提供有价值的新信息。因此，这项工作应该加强我们对正常听力过程的分子基础的理解。叙述：这项拟议的工作调查了导致耳聋的肌动蛋白突变对肌动蛋白功能的影响。这些结果将提供对这些突变导致耳聋的分子基础的洞察，以及对控制人类正常听力的分子机制的洞察。

DESCRIPTION (provided by applicant): The hair cell, located in the inner ear, is an essential component of the hearing process. The actin cytoskeleton plays a major role in hair cell function, through both the actin filament bundle in the stereocilium and the actin web in the cuticular plate. Six mutations have been found in gamma nonmuscle actin (DFNA 20/26) that cause autosomal dominant non-syndromic hearing loss. However, the molecular basis for the deafness caused by these mutations is unknown, in large part because of the inability to secure enough of the mutant actins to assess the effects of the mutations on actin function at the biochemical level. To address this problem and to understand the biochemical consequences of these mutations, we have cloned each mutation into yeast actin which is 91% identical to human gamma nonmuscle actin, and we can purify sufficient quantities of each of the mutant actins for biochemical analysis. These mutations cause allele-specific effects in yeast, but only one of the mutations affects polymerization of pure actin in vitro. This result suggest that the mutations may interfere, instead with actin's ability to be controlled by different actin filament regulatory proteins. We will use well-established biochemical and fluorescence microscopic assays of actin polymerization to assess the effects of these six deafness-causing mutations on actin's interaction with actin-binding proteins likely to to regulate actin filament function in the hair cell. Specifically, we will continue to use the yeast actin system to examine the mutations' effects on cofilin, an actin filament severing protein, Arp2/3 complex and formins which initiate actin polymerization and control actin filament length. We will examine the mutations' effects on actin's ability to interact with three deafness-associated actin filament bundling proteins, and espin, 1- catenin and Eps8. We will also use mass spectroscopy coupled with hydrogen-deuterium exchange to determine directly the effects of the mutations on actin filament conformation and flexibility. Finally, we will establish a baculovirus expression system to produce mammalian nonmuscle 3-isoactins containing the deafness mutations to allow assessment of the effects of the mutations in the actin in which they actually occur in the hair cell. RELEVANCE: Deafness is a major health problem in the United States. The proposed work will provide insight into the biochemical basis for the deafness caused by these six actin mutations. It will lead to a rigorous systematic examination of the properties of pure 3-nonmuscle isoactin. Additionally, the information gained on how these mutations affect actin's interaction with actin regulatory proteins will provide valuable new information concerning how actin functions and is regulated at the biochemical level in the normal ear. Thus, the work should enhance our understanding of the molecular basis of the normal hearing process. NARRATIVE: The proposed work investigates the effects on actin function of mutations in actin that lead to deafness. The results will provide insight into the molecular basis of the deafness these mutations cause as well as provide insight into the molecular mechanisms that govern normal hearing in humans.