USHERIN: STRUCTURAL AND FUNCTIONAL ANALYSIS

简介：Usher综合征是世界上导致聋哑/失明的主要遗传原因。遗传研究的进展揭示了这种疾病的高度异质性，九种特定基因被确定为三种临床亚型的病因。这些基因编码一系列产物，初步的功能分析指出了一些特定的相互作用，这表明存在一种功能性的引导蛋白“相互作用组”。“相互作用组”在体内的存在从未得到证实，而usher蛋白功能的关键亚细胞区室仍存在争议。各种usher基因的小鼠模型都显示出与角质层结构紊乱有关的立体纤毛发育缺陷，肌动蛋白副晶体数量异常，长度和形状变化，所有这些都与耳聋有关。没有usher小鼠模型出现视网膜变性，这阻碍了理解RP与usher综合征相关的进展。在本提案的初步结果中，我们证明了usher蛋白存在于气管上皮细胞的囊泡亚细胞部分中，并且确实在体内形成一个复合物，在大约50 svedberg的蔗糖梯度上沉积。对于三种不同的usher小鼠模型（迄今为止已经测试过的所有模型），我们发现在适应黑暗的动物暴露于光后，光感受器中的阻滞蛋白和转导蛋白易位存在缺陷。我们还在这些动物中证明了光诱导的光受体细胞退化，这表明usher小鼠确实具有健壮的视网膜表型，这在动物住房设施的环境中没有显示出来。这些发现构成了本提议的中心假设的基础，即引导蛋白的功能介导视网膜和耳蜗纤毛神经上皮细胞中蛋白质的易位。这一过程中的缺陷导致立体纤毛缺损和感光细胞变性。我们将在三个具体目标中检验这一假设。在第一个目的中，我们对纤毛气管上皮和视网膜提取物中的囊泡复合物进行生化分析，并对光感受器和毛细胞进行亚细胞定位实验。在第二个目标中，我们将检查所有已知的Usher综合征亚型的小鼠模型，以寻找蛋白质转位缺陷和光诱导的光感受器细胞变性，并在神经视网膜的器官型培养中直接使用囊泡运输抑制剂来验证这一假设。在第三个目标中，我们将通过阻断小鼠Corti器官培养中的囊泡运输和通过usher小鼠模型中usher蛋白的免疫组织学分析来研究囊泡运输在立体纤毛发育和维持中的作用。这些目标的成功完成将定义一个细胞过程，解释眼和耳纤毛神经上皮发育和功能缺陷，定义Usher综合征病理。这项工作将有助于协调该领域并促进旨在了解特定疾病机制的工作的加速，这是朝着开发有效治疗策略迈出的重要的第一步。Usher综合征是导致耳聋/失明的主要原因，并导致眼睛和视网膜感觉细胞的破坏。九种不同的基因已经被识别出来，编码的蛋白质相互作用，但与听觉和视觉的功能联系仍然未知。这一提议验证了一种假设，即功能连接是一个被称为蛋白质易位的过程，这可能会查明导致Usher患者耳聋和失明的缺陷过程。

DESCRIPTION: Usher syndrome is the leading genetic cause of deaf/blindness in the world. Progress in genetic research has revealed a high degree of heterogeneity for the disorder, with nine specific genes identified as causal for the three clinical sub-types. The genes encode an array of products and preliminary functional analysis points to a number of specific interactions that suggest the existence of a functional usher protein "interactome". The existence of the "interactome" in vivo has never been proven, and the critical subcellular compartments of usher protein function are still controversial. Mouse models for the various usher genes all display a developmental defect in stereocilia associated with disorganized structure in the cuticular plate, abnormal numbers of actin paracrystals, and variable lengths and shapes, all associated with deafness. None of the usher mouse models develop retinal degeneration, which has hampered progress in understanding RP associated with Usher syndrome. In the preliminary results of this proposal we demonstrate that the usher proteins are present in the vesicular sub-cellular fraction of tracheal epithelial cells and do indeed form a complex in vivo that sediments on sucrose gradients at around 50 Svedbergs. For three different usher mouse models (all that have been tested thus far) we show a defect in arrestin and transducin protein translocation in photoreceptors following exposure to light in dark adapted animals. We also demonstrate light-induced photo- receptor cell degeneration in these same animals, suggesting that the usher mice do indeed possess a robust retinal phenotype that is not revealed in the environment of animal housing facilities. These findings form the foundation for the central hypothesis of this proposal that usher proteins function to mediate translocation of proteins in ciliated neuroepithelial cells of the retina and the cochlea. Defects in this process lead to stereo- ciliary defects and photoreceptor cell degeneration. We will test this hypothesis in three specific aims. In the first aim we perform biochemical analysis of the vesicular complex in ciliated tracheal epithelium and retinal extracts, and perform subcellular localization experiments in photoreceptors and hair cells. In the second aim we will examine mouse models for all of the known subtypes of Usher syndrome for defects in protein trans- location and light induced photoreceptor cell degeneration and test the hypothesis directly using vesicle transport inhibitors in organotypic cultures of neural retina. In the third aim we will examine the role of vesicular transport in stereociliary development and maintenance by blocking vesicle transport in cultures of mouse organ of Corti and by immunohistological analysis of usher proteins in usher mouse models. Successful completion of these aims will define a cellular process that explains the defective development and function of ciliated neuroepithelium in the eye and ear that define Usher syndrome pathology. This work will help align the field and promote an acceleration of work aimed at understanding the specific disease mechanism, which is an essential first step towards the development of effective therapeutic strategies. PUBLIC HEALTH RELEVANCE Usher syndrome is the leading cause of deaf/blindness, and results in the destruction of sensory cells in the eye and the retina. Nine different genes have been identified, and the encoded proteins interact with each other, but the functional connection with hearing and vision is still unknown. This proposal tests the hypothesis that the functional connection is a process called protein translocation, which may pinpoint the defective process that causes deafness and blindness in Usher patients.