Ciliary pcoket matrix in photoreceptor health

睫状囊基质对光感受器健康的影响

基本信息

批准号：
9913548
负责人：
HUAIYU HU
金额：
$ 40.5万
依托单位：
UPSTATE MEDICAL UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-08-01 至 2023-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9913548
关键词：
Address Affinity Affinity Chromatography Atrophic Binding Binding Proteins Biochemical Biological Biological Assay Biological Models Blindness Cell Death Cell Surface Receptors Cessation of life Cilia Co-Immunoprecipitations Complex Cryoelectron Microscopy Data Disease ECM receptor Embryo Enzymes Epidermal Growth Factor Extracellular Matrix Extracellular Matrix Proteins Eye Eye Abnormalities Genes Genetic Diseases Goals Health Homologous Gene Homologous Protein Human Immunofluorescence Immunologic In Vitro Joubert syndrome Laminin Ligation Light Link Maintenance Mammals Mannose Meckel-Gruber syndrome Mediating Membrane Methods Modeling Molecular Mus Muscle eye brain disease Mutation Pathogenicity Pathway interactions Patients Photoreceptors Polysaccharides Process Proteins Proteomics Public Health Research Retina Retinal Degeneration Retinitis Pigmentosa Rodent Role Stains Structure Syndrome System Testing Therapeutic Intervention Transferase Vision Zebrafish age related alpha Dystroglycan brain malformation ciliopathy congenital muscular dystrophy effective therapy experimental study genetic manipulation glycosylation in vivo inherited retinal degeneration insight interdisciplinary approach member novel null mutation photoreceptor degeneration prevent protein complex recessive genetic trait targeted treatment

Address Affinity Affinity Chromatography Atrophic Binding Binding Proteins Biochemical Biological Biological Assay Biological Models Blindness Cell Death Cell Surface Receptors Cessation of life Cilia Co-Immunoprecipitations Complex Cryoelectron Microscopy Data Disease ECM receptor Embryo Enzymes Epidermal Growth Factor Extracellular Matrix Extracellular Matrix Proteins Eye Eye Abnormalities Genes Genetic Diseases Goals Health Homologous Gene Homologous Protein Human Immunofluorescence Immunologic In Vitro Joubert syndrome Laminin Ligation Light Link Maintenance Mammals Mannose Meckel-Gruber syndrome Mediating Membrane Methods Modeling Molecular Mus Muscle eye brain disease Mutation Pathogenicity Pathway interactions Patients Photoreceptors Polysaccharides Process Proteins Proteomics Public Health Research Retina Retinal Degeneration Retinitis Pigmentosa Rodent Role Stains Structure Syndrome System Testing Therapeutic Intervention Transferase Vision Zebrafish age related alpha Dystroglycan brain malformation ciliopathy congenital muscular dystrophy effective therapy experimental study genetic manipulation glycosylation in vivo inherited retinal degeneration insight interdisciplinary approach member novel null mutation photoreceptor degeneration prevent protein complex recessive genetic trait targeted treatment

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项目摘要

Retinitis pigmentosa 25 (RP25) is a common autosomal recessive genetic disease caused by abnormal eyes shut homolog protein (EYS). There is, however, little understanding of the mechanism by which mutations to EYS cause RP25 and there is no effective therapy. One of the challenges in developing a mechanistic un- derstanding of EYS function is the fact that the mouse does not have the EYS gene. To overcome this signifi- cant hurdle, we have developed a zebrafish model for RP25. Zebrafish express the EYS gene. EYS-deficient zebrafish show age-dependent photoreceptor degeneration similar to patients with RP25. Therefore, the goal of this project is to decipher the mechanisms of retinal degeneration caused by mutations in EYS using the zebrafish model system. EYS is a secreted protein containing multiple epidermal growth factor (EGF) and laminin globular (LG) domains. The latter is a conserved domain structure that is capable of binding to O- mannosyl glycans of α-dystroglycan, a cell surface receptor for extracellular matrix. Preliminary data indicate that the EYS protein binds to O-mannosyl glycans and is co-localized with the tectonic complex near the con- necting cilium/transition zone in photoreceptor cells. Abnormal O-mannosyl glycosylation underlie syndromic retinal atrophy found in congenital muscular dystrophies with brain malformations. Interestingly, hypomorphic mutations in one of the enzymes involved in O-mannosyl glycosylation are found in retinitis pigmentosa 76 (RP76). Tectonic complex proteins are implicated in severe ciliopathies such as Joubert syndrome. Our cen- tral hypothesis is that EYS is a key member of the ciliary pocket matrix interacting with O-mannosyl glycans and the tectonic complex at the connecting cilium/transition zone. Aim 1: Determine the roles of EYS in maintaining photoreceptor health. Aim 2: Determine the roles of O-mannosyl glycans in photoreceptor survival. Aim 3: Characterize EYS interactions with the tectonic complex at connecting cilium/transition zone. Multidisciplinary approaches including immuno-EM, cryo-EM, immunohistological staining, genetic manipu- lations in zebrafish embryos, and biochemical experiments such as co-immunoprecipitation, proximity ligation assay, and proteomic methods will be employed to accomplish the aims. Together, the proposed research will provide insights on molecular pathogenic mechanisms of the disease, uncover how EYS regulates ciliary func- tions in photoreceptors, and reveal the proteins that interact with EYS at the ciliary pocket. It has the potential to link several forms of retinal degeneration into a common biological pathway where cell-extracellular matrix interactions mediated by EYS help maintain the ciliary pocket structural integrity to maintain photoreceptor survival. We, thus, will have a better understanding of the mechanisms of photoreceptor degeneration caused by abnormal EYS, which will provide new opportunities to develop therapeutic interventions.

色素性视网膜炎25（RP25）是一种常见常染色体隐性遗传疾病，由异常引起眼睛关闭同源蛋白（EYS）。但是，对突变的机制几乎没有理解 EYS会导致RP25，并且没有有效的疗法。开发机械性的挑战之一 EYS功能的阐述是小鼠没有EYS基因的事实。克服这一重要 CANT障碍，我们已经开发了RP25的斑马鱼模型。斑马鱼表达Eys基因。 EYS缺陷斑马鱼的年龄依赖性光感受器变性类似于RP25患者。因此，目标这个项目的是破译由Eys突变引起的残余变性机理斑马鱼模型系统。 EYS是一种分泌的蛋白质，含有多重表皮生长因子（EGF）和层粘连蛋白球状（LG）结构域。后者是能够与O-结合的组成域结构 α-dystroglycan的甘露糖基聚糖，一种细胞表面受体，用于细胞外基质。初步数据指示 Eys蛋白与o-甘露糖基聚糖结合，并与构造复合物共定位在光感受器细胞中为纤毛/过渡带。 O-甘露糖基糖基化异常的基础综合症在先天性肌肉营养不良中发现的视网膜萎缩患有脑畸形。有趣的是，肌肉在色素性视网膜炎76中发现了参与O-甘露糖基糖基化的一种突变（RP76）。构造复合物蛋白在诸如乔伯特综合征等严重的纤毛病中实施。我们的cen- TRAL假设是EYS是与O-甘露糖基聚糖相互作用的纤毛袖珍基质的关键成员以及连接纤毛/过渡区的构造复合物。 AIM 1：确定EYS在维持光感受器健康中的作用。 AIM 2：确定O-甘露糖基聚糖在感光体存活中的作用。 AIM 3：表征与连接纤毛/过渡区域的构造复合物相互作用的表征。多学科的方法，包括免疫EM，冷冻EM，免疫组织学染色，遗传性manipu- 斑马鱼胚胎和生化实验（例如共免疫沉淀，接近连接）的最新作品测定和蛋白质组学方法将被雇用以实现目标。拟议的研究将在一起提供有关该疾病分子致病机制的见解，发现Eys如何调节纤毛功能光感受器中的tions，并揭示与纤毛袋中EYS相互作用的蛋白质。它有潜力将几种形式的残留变性连接到一个常见的生物学途径，其中细胞 - 细胞基质基质 EYS介导的相互作用有助于维持睫状口袋结构完整性以保持光感受器生存。因此，我们将更好地理解引起的光感受器变性的机制由异常的EYS撰写，这将为开发治疗干预提供新的机会。