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(申请人提供)：纤毛在大多数真核细胞表面作为感觉装置，在器官发生和发育过程中的组织模式形成中发挥重要作用。纤毛组装通过鞭毛内运输(IFT)和感觉转导能力是高度保守的所有纤毛生物。近十年来，随着人类疾病基因定位克隆的快速发展，各种疾病，如常染色体显性遗传性多囊肾病(ADPKD)、Joubert综合征(JBST)、Bardet-Biedl综合征(BBS)、肾结核(NPHP)、Meckel-Gruber综合征(MKS)和常染色体隐性遗传性多囊肾病(ARPKD)等已被定性为纤毛疾病。纤毛功能的建立和维持显然对生物体的健康至关重要。与普遍存在的纤毛一致，许多纤毛疾病以综合征的形式出现，影响到多个器官，包括肾脏、肝脏、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之间的联系仍然知之甚少。酶催化的小分子GTP酶起着分子开关的作用，控制着基本的细胞过程，经常与人类的各种病理状况相关。来自其他实验室和我们实验室的研究表明，三种保守且功能不佳的ADP核糖化因子(ARL)小GTP酶，ARL3、ARL6和Ar13b，作为显著的纤毛开关，使人或小鼠易患纤毛疾病。最大的障碍是分别开启和关闭纤毛ARL的鸟核苷酸交换因子(GEF)和GTP酶激活蛋白(GAP)，以及ARL的效应因子尚未确定。在其他疾病中，如结节性硬化症，确定由TSC2间隙抑制的GTP酶对于理解疾病具有变革性，我们认为这里的相应知识将对理解纤毛疾病具有类似的戏剧性影响。由于纤毛通路和纤毛病变基因的高度保守，秀丽线虫已被建立为一种简单而有效的模型来表征纤毛病变蛋白在其天然细胞环境中的生理作用。在上个基础研究阶段，我们成功地建立了线虫模型来研究纤毛病ARL的作用。我们最近的数据表明，纤毛中的纤毛ARL很可能被组织成两个不同的功能模块，位于纤毛的迷宫倒置蛋白(Inv)区。一个功能模块包含ARL-13-ARL-3-NPHP-2-UNC-119，其中UNC-119和肾单位蛋白NPHP-2与ARL-13协同作用，而与ARL-3拮抗，调节纤毛发生。第二个基因含有ARL-6-ARL-13-BBSome，它可能通过调节纤毛感觉受体的适当定位来调节纤毛信号。我们的初步结果也支持纤毛病ARL的作用从蠕虫到哺乳动物细胞都高度保守。基于此，我们的中心假设是，三种纤毛病变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转导。我们将利用线虫来确定体内调节因子和功能，并利用哺乳动物系统来确定对人类纤毛疾病的适用性。具体目标1是确定含有ARL的蛋白质模块中每个组分的调节因子的类型，我们希望确定GEF、GAP或纤毛病ARL的效应因子；特殊目标2是确定ARL-13、NPHP-2和UNC-119是否以及如何协调INV室中IFT的完整性和/或轴线稳定性，以及ARL-3是否通过脱乙酰酶HDAC-6依赖的方式拮抗ARL-13-NPHP-22-UNC-119的作用；具体目的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组装以及随后感觉受体在Inv室的适当纤毛定位来协调纤毛信号。这些研究具有揭示纤毛生物学突破的巨大潜力，并将提供纤毛生物发生和感觉功能在其自然环境中如何调节的开创性信息，阐明纤毛疾病的病因，并可能为疾病诊断和治疗提供新的靶点。