USHERIN: STRUCTURAL AND FUNCTIONAL ANALYSIS

描述(申请人提供)：Usher综合征是导致耳聋和失明的主要遗传原因，占美国2万聋人和盲人的一半以上。在与Usher综合征相关的10个已知遗传基因座中，Usher型Ha(USH2a)是最常见的。患有USH2A的患者患有先天性高频感音神经性听力损失，并伴有进行性视网膜色素变性。负责USH2A的基因最近被发现，并被证明编码一种新的蛋白质。USH2A基因的概念性翻译可以预测细胞外基质蛋白或细胞表面受体。在这项提议的初步结果部分，我们表明USH2A蛋白，我们称为usherin，是一种丰富的基底膜蛋白，组织分布广泛，但不是无处不在的。Usherin在耳蜗膜和视网膜基底膜中都非常丰富。了解迎春素蛋白如何对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。这项提议的目的是为了确定引座素蛋白的结构和功能特性。包含该蛋白功能结构域的融合肽以及表达的全长重组蛋白将被用来确定基底膜蛋白与迎春素相互作用，以及它们与迎春素分子的哪个结构域(S)相互作用。我们提供的数据说明了用这种方法定义的IV型胶原/前列环素相互作用。我们将使用永生化小鼠来源的工业边缘细胞、视网膜色素上皮(RPE)细胞和内皮细胞来定义引座素/细胞表面受体的相互作用，并确定涉及的特定受体。使用这种方法，我们展示了RPE细胞上特定结构域的迎春素受体结合。融合多肽将被改造成含有氨基酸替换，这些氨基酸替换是在亚瑟综合征ILA型家庭中发现的错义突变造成的。这些多肽将被用于建立竞争结合分析，以确定这些突变对迎春素功能的影响。综合起来，这些研究将提供一个基本的理解，即这种新的基底膜蛋白如何对发现它的基底膜的结构和功能特性做出贡献。此外，这些研究将为了解与USH2A病理相关的具体变化提供分子基础。

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.