Function of CEP290 protein in retinal ciliopathies and normal photoreceptor structure and function.

项目摘要。Bardet-Biedl综合征(BBS)是一种遗传异质性疾病，其特征是 视网膜变性(RD)、肥胖、多指畸形、肾脏缺陷和生殖器缺陷。这项提案的重点是 一个与BBS相关的基因，290 kDa的中心体蛋白(CEP290)，目的是了解其作用 CEP290在视杆感觉纤毛的正常结构和功能中的表达及其机制 CEP290缺乏性视网膜病变的病理生理学研究之所以选择CEP290，是因为取决于等位基因 和背景，CEP290缺陷导致一系列不同的缺陷，都涉及RD。与大多数其他人不同 BBS基因产物中，CEP290蛋白不能形成稳定的BBS膜包膜复合体，但 它与其他BBS蛋白在功能和物理上相互作用。虽然CEP290已被提议成为一种 纤毛门卫或连接纤毛(CC)内的结构脚手架，目前该领域被划分为 CEP290在CC中的定位、结构和功能作用及其机制 对纤毛贩运的监管还没有得到很好的理解。具体目标是：1.CEP290年表 CEP290或BBS4介导的视网膜(BBS14)亚细胞定位及BBS蛋白定位 变性以验证CEP290定位于外周微管二联体的假说并阐明 CEP290的结构作用、超分辨成像技术、结构照明显微镜 将使用(SIM)和随机光学重建显微镜(STORM)。来评估这一假设 相互作用蛋白如BBS4和BBS8在CEP290介导的RD中的错误定位是 将跟踪CEP290的缺失、BBS4在研发过程中的本地化以及CEP290 BBS4介导的RD的定位。为了将定位错误与疾病随时间的进展联系起来，组织学， 光学相干层析成像(OCT)、透射电子显微镜(TEM)和视网膜电描记术 将使用(ERG)。2.CEP290的BBs相互作用结构域的鉴定。BBS4和BBS8是 BBB一些亚基都依赖于CEP290的存在来进行适当的定位。来检验这些假设 CEP290通过其N端SMC同源结构域与BBS4和BBS8相互作用，BBS4和BBS8 BBS8通过它们的四肽重复序列(TPR)结构域、建议的结构域或 在建议的相互作用区域内有和没有突变的全长蛋白质将在 将进行hRPE1细胞和适当的共定位和免疫共沉淀的测试。3. 体内表达CEP290结构域对视网膜变性的功能修复作用确定入口点 CEP290及其结构域在蛋白质错误定位中的活体作用CEP290突变小鼠将接受视网膜下注射 注射和电穿孔表达结构域和全长CEP290蛋白。 了解CEP290的功能和发病机制是理解CEP290的基础 BBS和其他视网膜纤毛疾病的病理生理机制，以及设计新的治疗方法。

Project Summary. Bardet-Biedl syndrome (BBS) is a genetically heterogeneous disorder characterized by retinal degeneration (RD), obesity, polydactyly, renal defects, and genital defects. This proposal focuses on one BBS-associated gene, Centrosomal protein of 290 kDa (CEP290), with the goal of understanding the role of CEP290 in the normal structure and function of the rod sensory cilium, and the mechanisms of pathophysiology in RD caused by CEP290 deficiency. CEP290 was chosen because depending on the allele and background, CEP290 defects cause an array of diverse defects, all involving RD. In contrast to most other BBS gene products, CEP290 protein does not form a stable part of the BBSome membrane coat complex, but it interacts functionally and physically with other BBS proteins. While CEP290 has been proposed to be a ciliary gate-keeper or a structural scaffold within the connecting cilium (CC), the field is currently divided on the localization and the structural and functional roles of CEP290 within the CC, and the mechanism for CEP290 regulation of ciliary trafficking is not well understood. The Specific Aims are: 1. Chronology of CEP290 (BBS14) Subcellular Localization and BBS Protein Localization in CEP290- or BBS4-Mediated Retinal Degeneration To test the hypothesis that CEP290 localizes peripherally to microtubule doublets and elucidate the structural role of CEP290, the superresolution imaging techniques, Structured Illumination Microscopy (SIM) and Stochastic Optical Reconstruction Microscopy (STORM) will be used. To assess the hypothesis that mislocalization of interacting proteins such as BBS4 and BBS8,in CEP290-mediated RD is the direct result of the absence of CEP290, the localization of BBS4 over the course of RD will be tracked as well as CEP290 localization in BBS4-mediated RD. To correlate mislocalization with disease progression over time, histology, Optical Coherence Tomography (OCT), Transmission Electron Microscopy (TEM), and electroretinography (ERG) will be used. 2. Identification of the BBSome Interacting Domains of CEP290. BBS4 and BBS8 are BBSome subunits that both depend on the presence of CEP290 for proper localization. To test the hypotheses that CEP290 interacts with BBS4 and BBS8 through its N-terminal SMC homology domain, and that BBS4 and BBS8 interact with CEP290 through their tetratricopeptide repeats (TPR) domains, the proposed domains, or full length proteins with and without mutations within the proposed interaction domains, will be expressed in hRPE1 cells and tests for both proper co-localization and co-immunoprecipitation will be performed. 3. Functional Rescue of Retinal Degeneration with In Vivo CEP290 domain Expression. To determine the in vivo role of CEP290 and its domains in protein mislocalization, CEP290 mutant mice will receive sub-retinal injections and electroporation of expression constructs for rescue domains and full-length CEP290 protein. Understanding the functions and disease mechanisms of CEP290 is essential for understanding the pathophysiological mechanisms of BBS and other retinal ciliopathies, and designing new therapies.