USHERIN: STRUCTURAL AND FUNCTIONAL ANALYSIS

描述（由申请人提供）：Usher综合征是耳聋和失明的主要遗传原因，占美国20，000名聋哑人和盲人的一半以上。在10个已知的与Usher综合征相关的遗传位点中，Usher Ha型（USH 2A）是最常见的。USH 2A患者患有与进行性视网膜色素变性相关的先天性高频感音神经性听力损失。USH 2A的基因最近被鉴定出来，并显示出编码一种新的蛋白质。USH 2A基因的概念翻译预测细胞外基质蛋白或细胞表面受体。在本提案的初步结果部分，我们表明USH 2A蛋白，我们称之为usherin，是一种丰富的基底膜蛋白，具有广泛的，但不是无处不在的组织分布。Usherin在耳蜗和视网膜基底膜中都非常丰富。了解usherin蛋白如何对发现它的基底膜的结构架构和功能动力学做出贡献，将有助于我们破译缺乏这种蛋白的人的发病机制。该提案的目的是确定usherin蛋白的结构和功能特性。包含蛋白质功能结构域的融合肽以及表达的全长重组蛋白将用于鉴定什么样的基底膜蛋白与usherin相互作用，以及它们与usherin分子的什么结构域相互作用。我们目前的数据说明使用这种方法定义的IV型胶原/usherin相互作用。我们将采用永生小鼠衍生的视网膜边缘细胞，视网膜色素上皮（RPE）细胞，和内皮细胞来定义usherin/细胞表面受体的相互作用，并确定所涉及的特定受体。使用这种方法，我们证明结构域特异性usherin受体结合的RPE细胞。融合肽将被工程化，其具有由Ila型Usher综合征家族中发现的错义突变引起的氨基酸取代。这些肽将用于已建立的竞争性结合测定，以确定这些突变对usherin功能的影响。结合这些研究，将提供一个基本的理解，这类新的基底膜蛋白如何有助于其所在的基底膜的结构和功能特性，此外，这些研究将提供一个分子基础，了解与USH 2A病理相关的具体变化。

DESCRIPTION (provided by applicant): Usher syndrome is the leading hereditary cause of combined deafness and blindness, accounting for over half of the 20,000 deaf and blind people in the United States. Of the 10 known genetic loci associated with Usher syndrome, Usher type Ha (USH2A) is the most common. People with USH2A have congenital high frequency sensorineural hearing loss associated with progressive retinitis pigmentosa. The gene responsible for USH2A has recently been identified, and shown to encode a novel protein. Conceptual translation of the USH2A gene predicted either an extracellular matrix protein or cell surface receptor. In the preliminary results section of this proposal we show that the USH2A protein, which we call usherin, is an abundant basement membrane protein with widespread, but not ubiquitous, tissue distribution. Usherin is very abundant in both cochlear and retinal basement membranes. Understanding how the usherin protein contributes to the structural architecture and the functional dynamics of the basement membranes where it is found will help us decipher the mechanism of pathogenesis in people who lack this protein. The aims of this proposal are designed to identify the structural and functional properties of the usherin protein. Fusion peptides comprising the functional domains of the protein as well as an expressed full-length recombinant protein will be employed to identify what basement membrane proteins interact with usherin, and what domain(s) of the usherin molecule they interact with. We present data illustrating a type IV collagen/usherin interaction defined using this approach. We will employ immortomouse-derived strial marginal cells, retinal pigment epithelial (RPE) cells, and endothelial cells to define usherin/cell surface receptor interactions, and identify the specific receptors involved. Using this approach, we demonstrate domain-specific usherin receptor binding on RPE cells. Fusion peptides will be engineered that harbor amino acid substitutions resulting from missense mutations found in families with Usher syndrome type ila. These peptides will be employed in established competitive binding assays to determine the consequence of these mutations on usherin function. Combined, these studies will provide a basic understanding of how this new class of basement membrane protein contributes to the structural and functional properties of the basement membranes where it is found. Furthermore, these studies will provide a molecular basis for understanding the specific changes associated with USH2A pathology.