USHERIN: STRUCTURAL AND FUNCTIONAL ANALYSIS

描述（由申请人提供）：亚瑟综合症是导致耳聋和失明的主要原因，占美国 20,000 名聋哑人和盲人的一半以上。在与 Usher 综合征相关的 10 个已知遗传位点中，Usher Ha 型 (USH2A) 是最常见的。 USH2A 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。最近已鉴定出负责 USH2A 的基因，并显示其编码一种新蛋白质。 USH2A 基因的概念翻译预测了细胞外基质蛋白或细胞表面受体。在该提案的初步结果部分，我们表明 USH2A 蛋白（我们称之为 usherin）是一种丰富的基底膜蛋白，具有广泛但并非普遍存在的组织分布。 Usherin 在耳蜗和视网膜基底膜中含量非常丰富。了解导向蛋白如何影响其所在基底膜的结构架构和功能动态，将有助于我们破译缺乏这种蛋白质的人的发病机制。该提案的目的是确定迎来蛋白的结构和功能特性。包含蛋白质功能结构域以及表达的全长重组蛋白的融合肽将用于鉴定哪些基底膜蛋白与引座蛋白相互作用，以及它们与引座蛋白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 型胶原蛋白/引导蛋白相互作用。我们将采用永生小鼠来源的纹状体边缘细胞、视网膜色素上皮 (RPE) 细胞和内皮细胞来定义 usherin/细胞表面受体相互作用，并识别所涉及的特定受体。使用这种方法，我们证明了 RPE 细胞上的域特异性 usherin 受体结合。融合肽将被设计为包含由在具有IIa型亚瑟综合症的家族中发现的错义突变所产生的氨基酸取代。这些肽将用于已建立的竞争性结合测定中，以确定这些突变对引导蛋白功能的影响。结合起来，这些研究将提供对这种新型基底膜蛋白如何影响其所在基底膜的结构和功能特性的基本了解。此外，这些研究将为理解与 USH2A 病理学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.