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，其特征在于： 视网膜变性（RD）、肥胖、多指（趾）畸形、肾缺陷和生殖缺陷。该提案的重点是 一个BBS相关基因，290 kDa的中心体蛋白（CEP 290），目的是了解其作用 CEP 290在视杆感觉纤毛的正常结构和功能中的作用，以及CEP 290的作用机制。 CEP 290缺乏引起RD的病理生理学。选择CEP 290是因为取决于等位基因 在背景技术中，CEP 290缺陷导致一系列不同的缺陷，所有这些缺陷都涉及RD。与大多数其他 BBS基因产物、CEP 290蛋白并不构成BBSome膜被复合物的稳定部分，但 它在功能上和物理上与其它BBS蛋白相互作用。虽然CEP 290已被提议为 睫状体看门人或连接纤毛（CC）内的结构支架，该领域目前分为 CEP 290在CC中的定位、结构和功能，以及CEP 290在CC中的作用机制。 对纤毛运输的调节还没有很好的理解。具体目标是：1。CEP 290年表 （BBS 14）在CEP 290或BBS 4介导的视网膜中的亚细胞定位和BBS蛋白定位 为了验证CEP 290定位于微管偶联体外周的假设，并阐明 CEP 290的结构作用，超分辨成像技术，结构照明显微术 (SIM)和随机光学重建显微镜（STORM）。为了评估假设， 在CEP 290介导的RD中，相互作用蛋白如BBS 4和BBS 8的错误定位是以下因素的直接结果： 如果没有CEP 290，将跟踪研发过程中BBS 4的定位以及CEP 290 定位于BBS 4介导的RD。为了将错误定位与随时间推移的疾病进展相关联，组织学， 光学相干断层扫描（OCT）、透射电子显微镜（TEM）和视网膜电图 (ERG)将用于2. CEP 290的BBSome相互作用结构域的鉴定。BBS 4和BBS 8 BB一些亚基，它们都依赖于CEP 290的存在来进行适当的定位。为了验证假设 CEP 290通过其N-末端SMC同源结构域与BBS 4和BBS 8相互作用，而BBS 4和 BBS 8通过它们的三肽重复序列（TPR）结构域与CEP 290相互作用，所提出的结构域，或 在所提出的相互作用结构域内具有和不具有突变的全长蛋白质将在 将进行hRPE 1细胞和适当共定位和免疫共沉淀试验。3. 用体内CEP 290结构域表达功能性拯救视网膜变性。为了确定 CEP 290及其结构域在蛋白质错误定位中的体内作用，CEP 290突变小鼠将接受视网膜下 注射和电穿孔用于拯救结构域和全长CEP 290蛋白的表达构建体。 了解CEP 290的功能和疾病机制对于理解 BBS和其他视网膜睫状体病的病理生理机制，并设计新的治疗方法。

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.