MEMBRANE FUSION IN RETINAL ROD OUTER SEGMENTS

描述（由申请人提供）：视网膜退行性疾病的特征通常是感光体结构异常。我们对感光细胞健康和疾病的理解的核心是关于感光细胞外节如何形成以及哪些过程有助于其结构稳定性的基本知识。由rds基因编码的Peripherin/rds（p/rds）对外节的形成、组织和维持至关重要。在人类中，该基因内超过150个突变中的任何一个都会导致以异常感光器结构为特征的多种迟发性进行性视网膜营养不良。p/rds介导的视网膜营养不良的致病机制尚不清楚，然而，基于我们最近的结构研究，我们对p/rds功能的理解获得了新的见解，该研究鉴定了该蛋白的C-末端结构域与称为固有无序结构域（IDD）的结构同源.异常大量的含有IDD的蛋白质与人类疾病相关，包括癌症、心血管疾病、淀粉样变性、神经变性疾病和糖尿病。IDD是通过与一个或几个结合配偶体相互作用而稳定的随机结构。结构灵活性赋予蛋白质多功能性，结合配偶体充当功能调节剂。我们预计，破译的IDD C-末端结构域的p/rds的功能是至关重要的理解的光感受器中的OS形成的机制，以及如何在该地区的突变有助于杆或锥显性营养不良。因此，本项目的主要目标是测试IDD p/rds C-末端结构域在视杆OS的生物发生和稳定OS结构中起直接作用的假设。我们将定义p/rds的C-末端结构域在OS形态发生和稳定性中的作用，并了解二元（直接）蛋白质与该结构域结合的生理意义。一个全面的战略，涉及在体内小鼠模型和体外生物物理方法的建议。在具体目标#1a中，使用体内电穿孔将遗传修饰的rds引入发育中的小鼠视网膜中，我们将确定p/rds C-末端的截短和功能突变如何调节p/rds运输以及OS结构的建立和维持。该目的的主要目的是绘制小鼠中的p/rds定位信号。此外，我们将在体内观察p/rds与其已知蛋白质结合配偶体之间的相互作用，并使用双分子荧光互补测定法评估这些相互作用的抑制如何影响OS形成。此外，我们将评估疾病相关的p/rds C末端突变如何改变p/rds靶向、组装和结构维持，并将这些突变体中固有无序的C末端结构域的折叠如何改变结合伴侣亲和力相关联。这些研究有望为外周蛋白/rds的功能提供重要的机制见解，并更好地理解视网膜的正常生物学和与p/rds突变相关的视网膜变性。了解p/rds在光感受器外节的建立和维持中的功能，将为全面了解四跨膜蛋白的功能以及内在无序结构域在细胞过程和疾病发病机制中的作用提供线索。这些研究将为治疗迟发性视网膜变性的治疗策略的设计提供基础，这是老龄化人口中日益严重的问题。公共卫生相关性：光感受器外周蛋白/rds的突变是老年人失明和视力受损的最常见原因之一。具体而言，它们是美国和北方常染色体显性视网膜色素变性（adRP）的最普遍原因;该基因内的超过150个突变导致以异常光感受器结构为特征的多种迟发性进行性视网膜营养不良。我们将确定外周蛋白/rds突变如何有助于光感受器功能障碍，通过研究这种蛋白质的结构独特区域在光感受器形成中的作用，使用体外方法和人类疾病的小鼠模型。我们的研究将揭示外周蛋白/rds介导的视网膜营养不良的发病机制的新见解，并为治疗这些疾病的治疗策略的发展奠定基础。

DESCRIPTION (provided by applicant): Retinal degenerative diseases are often characterized by abnormalities in photoreceptor structure. Central to our understanding of photoreceptor health and disease is a fundamental knowledge regarding how photoreceptor outer segments are formed and what processes contribute to their structural stability. Peripherin/rds (p/rds), encoded by the rds gene, is essential for the formation, organization and maintenance of outer segments. In humans, any one of over 150 mutations within this gene results in a broad variety of late onset progressive retinal dystrophies characterized by abnormal photoreceptor structure. The pathogenic mechanism(s) underlying p/rds mediated retinal dystrophies are unknown, however new insight has been gained in our understanding of p/rds function based upon our recent structural studies identifying the C- terminal domain of this protein as homologous to structures called intrinsically disordered domains (IDD). An unusually large number of IDD containing proteins are associated with human diseases, including cancer, cardiovascular disease, amyloidosis, neurodegenerative diseases and diabetes. IDDs are random structures which are stabilized through interactions with one or with several binding partners. Structural flexibility imparts multi-functionality to the protein and binding partners act as regulators of function. We anticipate that deciphering the function of the IDD C-terminal domain of p/rds is critical to understanding the mechanism of OS formation in photoreceptors and how mutations in this region contribute to rod or cone dominant dystrophies. Hence the primary goals of this project are to test the hypothesis that the IDD p/rds C-terminal domain plays a direct role in the biogenesis of rod OS and in stabilizing OS structure. We will define the role(s) of the C-terminal domain of p/rds in OS morphogenesis and stability and understand the physiological implications of binary (direct) protein binding to this domain. A comprehensive strategy involving in vivo mouse models and in vitro biophysical approaches is proposed. In specific Aim #1a using in vivo electroporation to introduce genetically modified rds into the developing mouse retina we will determine how truncation and functional mutations in the p/rds C-terminus modulate p/rds trafficking as well as the establishment and maintenance of OS structure. A primary objective of this aim is to map the p/rds localization signal in mouse. Furthermore, we will visualize the interaction between p/rds and its known protein binding partners in vivo and assess how inhibition of these interactions affects, OS formation using bi- molecular fluorescence complementation assays. In addition, we will assess how disease linked p/rds C- terminal mutations alter p/rds targeting, assembly and maintenance of structure and correlate how folding of the intrinsically disordered C-terminal domain in these mutants may alter binding partner affinity. The proposed studies are expected to provide significant mechanistic insights into peripherin/rds function and a better understanding of both normal biology of the retina and retinal degenerations associated with mutations in p/rds. An understanding of the function of p/rds in the establishment and maintenance of photoreceptor outer segments should provide clues to an overall understanding of tetraspanin protein function and the role of intrinsically disordered domains in cellular processes and disease pathogenesis. These studies will provide the underpinnings for the design of therapeutic strategies to treat late-onset retinal degenerations, a growing problem in an aging population. PUBLIC HEALTH RELEVANCE: Mutations in photoreceptor peripherin/rds are one of the most common causes of blindness and impaired vision in the elderly. Specifically, they are the most prevalent cause of autosomal dominant retinitis pigmentosa (adRP) in the United States and Northern Europe; over 150 mutations within this gene result in a broad variety of late onset progressive retinal dystrophies characterized by abnormal photoreceptor structure. We will determine how peripherin/rds mutations contribute to photoreceptor dysfunction by examining the role of structurally unique regions of this protein in photoreceptor formation using in vitro methods and mouse models of human disease. Our studies should reveal novel insights into the pathogenesis of peripherin/rds mediated retinal dystrophies and form the underpinnings for the development of therapeutic strategies to treat these diseases.