Biochemical Consequences of Deafness-causing Actin Mutations

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，它通过立体纤毛中的肌动蛋白细丝束。 以及角质板中的肌动蛋白网。已在伽马非肌肉肌动蛋白(DFNA)中发现六种突变 20/26)，可导致常染色体显性遗传性非综合征性听力损失。然而，分子基础上的 这些突变导致的耳聋是未知的，很大程度上是因为无法确保足够的 突变的肌动蛋白在生化水平上评估突变对肌动蛋白功能的影响。至 为了解决这个问题并了解这些突变的生化后果，我们克隆了 每个突变都转化成酵母肌动蛋白，它与人类伽马非肌肉肌动蛋白有91%的同源性，我们可以提纯 足够数量的每个突变肌动蛋白用于生化分析。这些突变导致等位基因 在酵母中的影响，但只有一个突变影响纯肌动蛋白的体外聚合。这个结果 提示突变可能会干扰肌动蛋白受不同肌动蛋白细丝控制的能力 调节蛋白。我们将使用成熟的生物化学和荧光显微镜分析肌动蛋白 聚合反应评估这六个导致耳聋的突变对肌动蛋白与放线蛋白结合的相互作用的影响 可能调节毛细胞中肌动蛋白细丝功能的蛋白质。具体来说，我们将研究 突变对Cofilin的影响，Cofilin是一种肌动蛋白切割蛋白，肌球蛋白交易肌动蛋白单体和 其他启动肌动蛋白聚合和控制的蛋白质位于立体纤毛、Arp2/3复合体和福尔马林上 肌动蛋白细丝长度和ESpin，它使立体纤毛束中的肌动蛋白细丝发生交联。我们会 也可以使用质谱学和氢-氚交换来直接确定 肌动蛋白细丝构象和柔韧性上的突变。相关性：耳聋是一种主要的健康问题 在美国的问题。拟议的工作将提供对生物化学基础的见解 由这六种肌动蛋白突变引起的耳聋。此外，获得的关于这些突变是如何 影响肌动蛋白与肌动蛋白调节蛋白的相互作用将提供关于如何 在正常耳朵中，肌动蛋白的功能和调节处于生化水平。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。

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 allelespecific 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 actinbinding proteins likely to to regulate actin filament function in the hair cell. Specifically, we will examine the mutations' effects on cofilin, an actin.filament severing protein, myosin which trafficks actin monomers and other proteins up the stereocilium, Arp2/3 complex and formins which initiate actin polymerization and control actin filament length, and espin which crosslinks actin filaments in the bundle of the stereocilium. 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. 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. 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.