USHERIN: STRUCTURAL AND FUNCTIONAL ANALYSIS

描述（由申请人提供）：Usher综合征是导致耳聋和失明的主要遗传原因，占美国2万聋哑人和盲人的一半以上。在已知的10个与Usher综合征相关的基因位点中，Usher型Ha （USH2A）最为常见。患有USH2A的人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的基因最近被确定，并被证明编码一种新的蛋白质。USH2A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USH2A蛋白，我们称之为引子蛋白，是一种丰富的基底膜蛋白，具有广泛的组织分布，但不是普遍存在的。引导素在耳蜗和视网膜基底膜中都非常丰富。了解引子蛋白如何影响基底膜的结构结构和功能动力学，将有助于我们破译缺乏这种蛋白质的人的发病机制。本提案的目的是确定引子蛋白的结构和功能特性。融合肽包括蛋白质的功能域以及表达的全长重组蛋白，将用于确定哪些基底膜蛋白与引导素相互作用，以及它们与引导素分子的哪些结构域相互作用。我们提供的数据说明了使用这种方法定义的IV型胶原/引导蛋白相互作用。我们将使用永不朽小鼠来源的纹状体边缘细胞、视网膜色素上皮细胞（RPE）和内皮细胞来定义引导素/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，我们证明了区域特异性引导受体在RPE细胞上的结合。融合肽将被设计成含有在Usher综合征ila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试验，以确定这些突变对引导功能的影响。综上所述，这些研究将提供一个基本的理解，这类新的基膜蛋白是如何促进基膜的结构和功能特性的。此外，这些研究将为了解与USH2A病理相关的特定变化提供分子基础。

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.