Biochemical consequences of Deafness-causing actin mutations

描述（由申请人提供）：毛细胞位于内耳，是听力过程的重要组成部分。肌动蛋白细胞骨架通过体纤毛中的肌动蛋白丝束和角质层板中的肌动蛋白网在毛细胞功能中起重要作用。在γ -非肌动蛋白（DFNA 20/26）中发现了6个突变，可导致常染色体显性非综合征性听力损失。然而，由这些突变引起的耳聋的分子基础尚不清楚，这在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，它与人类γ非肌肉肌动蛋白相同91%，我们可以纯化足够数量的每种突变肌动蛋白用于生化分析。这些突变在酵母中引起等位基因特异性效应，但只有一种突变影响体外纯肌动蛋白的聚合。这一结果表明，突变可能会干扰肌动蛋白受不同肌动蛋白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致聋突变对肌动蛋白与肌动蛋白结合蛋白相互作用的影响，这些蛋白可能调节毛细胞中肌动蛋白丝的功能。具体来说，我们将继续使用酵母肌动蛋白系统来检测突变对cofilin的影响，cofilin是一种肌动蛋白丝切断蛋白，Arp2/3复合物和formins启动肌动蛋白聚合和控制肌动蛋白丝长度。我们将研究突变对肌动蛋白与三种耳聋相关肌动蛋白丝束蛋白、espin、1- catenin和Eps8相互作用能力的影响。我们还将使用质谱结合氢-氘交换来直接确定突变对肌动蛋白丝构象和柔韧性的影响。最后，我们将建立一个杆状病毒表达系统，以产生含有耳聋突变的哺乳动物非肌肉3-异肌动蛋白，以评估它们在毛细胞中实际发生的肌动蛋白突变的影响。相关性：耳聋在美国是一个主要的健康问题。这项工作将为这六种肌动蛋白突变引起的耳聋的生化基础提供见解。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的相互作用的信息将提供有关肌动蛋白在正常耳中如何在生化水平上发挥作用和调节的有价值的新信息。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。本研究旨在研究导致耳聋的肌动蛋白突变对肌动蛋白功能的影响。这些结果将为这些突变引起的耳聋的分子基础以及控制人类正常听力的分子机制提供见解。

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.