Exploration of the functions of the ciliopathy Arls in cilia

纤毛病Arls在纤毛中的功能探讨

• 批准号: 9204826
• 负责人: Jinghua Hu
• 金额: $ 35.78万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-01-15 至 2019-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9204826
• 关键词: ADP-Ribosylation Factors; ADP-ribosylation factor 6; ARL3 gene; Address; Affect; Arl proteins; Autosomal Dominant Polycystic Kidney; Autosomal Recessive Polycystic Kidney; Bardet-Biedl Syndrome; Biology; Caenorhabditis elegans; Cell physiology; Cell surface; Cilia; Complex; Data; Deacetylase; Defect; Development; Devices; Disease; Environment; Etiology; Eukaryotic Cell; Eye; Fatty acid glycerol esters; Funding; GTPase-Activating Proteins; Genes; Genetic Models; Goals; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate Phosphohydrolases; Hereditary Disease; Histone Deacetylase; Human; Human Genetics; Human Pathology; Joubert syndrome; Kidney; Knowledge; Laboratories; Light; Limb structure; Liver; Maintenance; Mammalian Cell; Modeling; Molecular; Monomeric GTP-Binding Proteins; Mus; Nephronophthisis; Neuraxis; Organ; Organism; Organogenesis; Pathogenesis; Pathologic; Pathology; Pathway interactions; Pattern Formation; Phenotype; Physiological; Play; Polycystic Kidney Diseases; Process; Property; Proteins; Research; Resources; Role; Seminal; Sensory; Sensory Receptors; Signal Transduction; Syndrome; System; TSC2 gene; Therapeutic Intervention; Tissues; Tuberous Sclerosis; Work; base; ciliopathy; cilium biogenesis; clinically relevant; design; disease diagnosis; gene function; human disease; in vivo; insight; novel; positional cloning; protein complex; public health relevance; receptor; therapeutic target

 DESCRIPTION (provided by applicant): Cilia serve as sensory devices on most eukaryotic cell surfaces and play essential roles in organogenesis and tissue pattern formation during development. Ciliary assembly via intraflagellar transport (IFT) and sensory transduction capabilities are highly conserved in all ciliated organisms. With rapid advancements in the positional cloning of human disease genes in the past decade, a wide variety of disorders, such as autosomal dominant polycystic kidney disease (ADPKD), Joubert syndrome (JBST), Bardet-Biedl syndrome (BBS), nephronophthisis (NPHP), Meckel-Gruber syndrome (MKS), and autosomal recessive polycystic kidney disease (ARPKD), have been characterized molecularly as ciliopathies. The establishment and maintenance of ciliary function are clearly essential for the well-being of an organism. Consistent with the ubiquitous presence of cilia, many ciliopathies occur as syndromic disorders that affect multiple organs, including the kidneys, liver limbs, eyes, central nervous system (CNS), and fat storage tissue. Despite the physiological and clinical relevance of cilia, the core machinery that regulates cilia biogenesis and function as well as the connection between the disease gene function and pathology remain poorly understood. Enzymatic small GTPases act as molecular switches, which control fundamental cellular processes and are often correlated with various human pathological conditions. Studies from other and our laboratories demonstrated that three conserved and poorly characterized ADP-ribosylation factor-like (ARL) small GTPases, ARL3, ARL6, and ARL13B, act as prominent ciliary switches, with disrupted function predisposing human or mice to ciliopathies. The paramount obstacle being that the guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) that switch ciliary ARLs on and off, respectively, and the effectors of ARLs have not been identified. In other disorders, such as tuberous sclerosis, identifying the GTPase inhibited by the TSC2 GAP was transformative in understanding the disease, and we propose that the corresponding knowledge here would have a similar dramatic effect on understanding ciliopathies. Due to highly conserved cilia pathways and ciliopathy genes, Caenorhabditis elegans has been established as a simple and effective model for characterizing the physiological roles of ciliopathy proteins in their native cellular environments. In last fundig period, we have successfully established C. elegans as a model to investigate the roles of ciliopathy ARLs. Our recent data suggested that ciliary ARLs are likely organized into two distinct functional modules in the enigmatic inversin (InV) compartment of cilia. One function module contains ARL-13-ARL-3-NPHP-2-UNC-119, in which UNC-119 and nephronophthisis protein NPHP-2 act synergistically with ARL-13, but antagonistically with ARL-3, in regulating ciliogenesis. The second one contains ARL-6-ARL-13-BBSome, which may regulate cilia signaling through regulating the proper localization of ciliary sensory receptors. Our preliminary results also supported that the roles of ciliopathy ARLs are highly conserved from worm to mammalian cells. Based on these, our central hypothesis is that the three ciliopathy ARLs and their regulators are organized into distinct complexes to coordinate cilia biogenesis and signaling, respectively. We will employ C. elegans to identify in vivo regulators and functions, and mammalian systems to determine the applicability to human ciliopathies. Specific Aim 1 is to characterized the type of regulators for each component in ARL-containing protein module, and we hope to identify GEFs, GAPs, or effectors for ciliopathy ARLs; Specific Aim 2 is to ascertain whether and how ARL-13, NPHP-2, and UNC-119 coordinate IFT integrity and/or axonemal stability in the InV compartment, and whether ARL-3 antagonizes the roles of ARL-13-NPHP-22-UNC-119 through deacetylase HDAC-6-dependent manner; Specific Aim 3 is to determine whether the ARL-6-ARL-13-BBSome module coordinates cilia signaling through the mechanism that ARL-13 promotes ARL-6 activation, BBSome-cargo assembly, and subsequent proper ciliary localization of sensory receptors in the InV compartment. The proposed studies have great potentials for unveiling breakthroughs in cilia biology, and would provide seminal information about how cilia biogenesis and sensory function are regulated in their native environment, shed light on the etiologies of ciliopathies, and potentially provide novel targets fo disease diagnosis and treatment.

 描述（由申请人提供）：纤毛作为大多数真核细胞表面的感觉装置，在发育过程中的器官发生和组织模式形成中发挥重要作用。通过鞭毛内运输（IFT）和感觉转导能力进行的纤毛组装在所有纤毛生物中都高度保守。过去十年，随着人类疾病基因定位克隆的快速进展，多种疾病，如常染色体显性多囊肾病（ADPKD）、Joubert综合征（JBST）、Bardet-Biedl综合征（BBS）、肾病（NPHP）、Meckel-Gruber综合征（MKS）和常染色体隐性多囊肾病 肾脏疾病（ARPKD）在分子水平上被定性为纤毛病。纤毛功能的建立和维持显然对于生物体的健康至关重要。与纤毛的普遍存在相一致，许多纤毛病作为影响多个器官的综合征性疾病发生，包括肾脏、肝脏四肢、眼睛、中枢神经系统 (CNS) 和脂肪储存组织。尽管纤毛具有生理和临床相关性，但调节纤毛生物发生和功能的核心机制是 以及疾病基因功能和病理学之间的联系仍然知之甚少。酶促小 GTP 酶充当分子开关，控制基本的细胞过程，并且通常与各种人类病理状况相关。其他实验室和我们实验室的研究表明，三种保守且特征不明确的 ADP-核糖基化因子样 (ARL) 小 GTP 酶 ARL3、ARL6 和 ARL13B 充当显着的纤毛开关，其功能破坏使人类或小鼠易患纤毛病。最重要的障碍是分别打开和关闭纤毛 ARL 的鸟嘌呤核苷酸交换因子 (GEF) 和 GTP 酶激活蛋白 (GAP) 以及 ARL 的效应器尚未确定。在其他疾病中，例如结节性硬化症，识别 TSC2 GAP 抑制的 GTP 酶对于理解该疾病具有变革意义，我们建议此处的相应知识对于理解纤毛病也将产生类似的显着效果。由于纤毛途径和纤毛病基因高度保守，秀丽隐杆线虫已被建立为一种简单有效的模型，用于表征纤毛病蛋白在其天然细胞环境中的生理作用。在上个fundig阶段，我们成功建立了秀丽隐杆线虫作为模型来研究纤毛病ARL的作用。我们最近的数据表明，纤毛 ARL 可能在纤毛神秘的反相 (InV) 区室中组织成两个不同的功能模块。其中一个功能模块包含ARL-13-ARL-3-NPHP-2-UNC-119，其中UNC-119和肾结核蛋白NPHP-2与ARL-13协同作用，但与ARL-3拮抗调节纤毛发生。第二种包含 ARL-6-ARL-13-BBSome，它可以通过调节纤毛感觉受体的正确定位来调节纤毛信号传导。我们的初步结果还支持纤毛病 ARL 的作用从线虫到哺乳动物细胞都高度保守。基于这些，我们的中心假设是，三种纤毛病 ARL 及其调节因子被组织成不同的复合体，分别协调纤毛的生物发生和信号传导。我们将利用秀丽隐杆线虫来鉴定体内调节因子和功能，并利用哺乳动物系统来确定其对人类纤毛病的适用性。具体目标1是表征包含ARL的蛋白质模块中每个成分的调节子类型，我们希望识别纤毛病ARL的GEF、GAP或效应子；具体目标2是确定ARL-13、NPHP-2和UNC-119是否以及如何协调InV区室中的IFT完整性和/或轴丝稳定性，以及ARL-3是否通过脱乙酰酶HDAC-6依赖性方式拮抗ARL-13-NPHP-22-UNC-119的作用；具体目标 3 是确定 ARL-6-ARL-13-BBSome 模块是否通过 ARL-13 促进 ARL-6 激活、BBSome-cargo 组装以及随后 InV 区室中感觉受体的正确纤毛定位的机制来协调纤毛信号传导。拟议的研究具有揭示纤毛生物学突破的巨大潜力，并将提供关于纤毛生物发生和感觉功能如何在其原生环境中调节的开创性信息，揭示纤毛病的病因学，并有可能为疾病诊断和治疗提供新的靶点。