Biochemical consequences of Deafness-causing actin mutations

导致耳聋的肌动蛋白突变的生化后果

• 批准号: 7850295
• 负责人: Peter A. Rubenstein
• 金额: $ 24.95万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-17 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7850295
• 关键词: Actin-Binding Protein; Actins; Address; Affect; Alleles; Baculovirus Expression System; Baculoviruses; Behavior; Binding; Biochemical; Biological Assay; Bundling; Cell physiology; Cells; Classification; Code; Collaborations; Complex; Coupled; Cytoskeleton; Defect; Deuterium; Disease; Ear; Exhibits; F-Actin; Filament; Fluorescence; Hair Cells; Health; Hearing; Human; Hydrogen; In Vitro; Internet; Isoactin; Kinetics; Label; Labyrinth; Lead; Length; Maintenance; Mass Spectrum Analysis; Microfilaments; Microscopic; Molecular; Molecular Conformation; Muscle; Mutation; Phenotype; Play; Plus End of the Actin Filament; Polymers; Process; Property; Protein Isoforms; Proteins; Recombinants; Relative (related person); Research; Role; Secure; Side; Solutions; Stereocilium; Surface; System; Testing; United States; Work; Yeasts; actin filament bundling proteins; base; cofilin; crosslink; deafness; early onset; flexibility; genetic regulatory protein; hearing impairment; insight; interest; monomer; mutant; polymerization; profilin

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.

描述（由申请人提供）：位于内耳的毛细胞是听力过程的重要组成部分。肌动蛋白细胞骨架通过静纤毛中的肌动蛋白丝束和表皮板中的肌动蛋白网在毛细胞功能中起主要作用。在γ-非肌肉肌动蛋白（DFNA 20/26）中发现了六种突变，导致常染色体显性遗传的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是未知的，在很大程度上是因为无法获得足够的突变肌动蛋白，以评估突变对肌动蛋白功能在生化水平上的影响。为了解决这个问题，并了解这些突变的生化后果，我们已经克隆到酵母肌动蛋白，这是91%相同的人γ非肌肉肌动蛋白的每个突变，我们可以纯化足够数量的每个突变肌动蛋白的生化分析。这些突变在酵母中引起等位基因特异性效应，但只有一个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，而不是与肌动蛋白的能力，由不同的肌动蛋白丝调控蛋白。我们将使用完善的生物化学和荧光显微镜测定肌动蛋白聚合，以评估这六个突变的影响，肌动蛋白的相互作用与肌动蛋白结合蛋白可能调节肌动蛋白丝功能的毛细胞。具体来说，我们将继续使用酵母肌动蛋白系统来检查突变对cofilin（一种肌动蛋白丝切断蛋白）、Arp 2/3复合物和formin（启动肌动蛋白聚合并控制肌动蛋白丝长度）的影响。我们将研究突变对肌动蛋白与三种与染色体相关的肌动蛋白丝捆绑蛋白，espin，1- catenin和Eps 8相互作用的能力的影响。我们还将使用质谱结合氢氘交换直接确定突变对肌动蛋白丝构象和灵活性的影响。最后，我们将建立一个杆状病毒表达系统，以产生哺乳动物非肌肉3-异肌动蛋白含有耳聋突变，允许评估的影响，在肌动蛋白中，他们实际上发生在毛细胞中的突变。相关性：耳聋是美国的一个主要健康问题。拟议的工作将提供深入了解这六个肌动蛋白突变引起的耳聋的生化基础。这将导致对纯3-非肌肉异肌动蛋白的性质进行严格的系统检查。此外，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如何在正常耳的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。旁白：这项工作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。这些结果将提供对这些突变导致的耳聋的分子基础的深入了解，并提供对人类正常听力的分子机制的深入了解。