Fleer: Functional study in regulation of vertebrate cilia tublin modification

Fleer：脊椎动物纤毛微管蛋白修饰调节的功能研究

• 批准号: 7650121
• 负责人: Narendra H Pathak
• 金额: $ 13.77万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7650121
• 关键词: Adolescent; Affinity Chromatography; Antibodies; Axon; Bardet-Biedl Syndrome; Biochemical; Biochemical Process; Biological Assay; C-terminal; Caenorhabditis elegans; Cell physiology; Cells; Centrioles; Chemicals; Chlamydomonas; Cilia; Co-Immunoprecipitations; Code; Coupled; Defect; Development; Disease; Dominant-Negative Mutation; Embryo; Enzymes; Epitopes; Family member; Functional disorder; Genes; Genetic; Homologous Gene; Immunofluorescence Immunologic; In Situ; In Situ Hybridization; Kidney Diseases; Kinesin; Larva; Link; Mass Spectrum Analysis; Mechanics; Mediating; Modeling; Modification; Motor; Multienzyme Complexes; Nephronophthisis; Organism; Pathway interactions; Pattern; Physiology; Play; Post-Translational Protein Processing; Process; Proteins; Regulation; Research; Role; Scaffolding Protein; Sensory; Specific qualifier value; Stimulus; Syndrome; Techniques; Testing; Tissues; Transgenic Organisms; Tubulin; Two-Hybrid System Techniques; Vertebrates; Yeasts; Zebrafish; base; career; cilium biogenesis; cilium motility; human disease; in vivo; kinetosome; mutant; novel; protein complex; public health relevance; relating to nervous system; scaffold; sensor; success; therapeutic target; tyrosyltubulin ligase; zebrafish genome

DESCRIPTION (provided by applicant): Human disease syndromes involving cystic nephropathies and sensori-neural deficits are linked to defects of cilia, for example, Senior-Loken syndrome, Bardet-Biedl Syndrome, Juvenile Nephronophthisis. Cilia in their stationary or motile forms serve both sensory and effector functions in cellular physiology. Genetic and biochemical analysis of ciliogenesis in simple organisms such as Chlamydomonas and C.elegans have led to detailed understanding of cellular processes invoved in cilia formation, however their significance in pathophysiology of ciliary disease necessitates studies in vertebrates. In characterization of the zebrafish cystic mutant fleer we identified widespread cilia defects that arise from reduction in glutamylation of axonemal tubulins. Polyglutamylation is a posttranslational modification detected on tubulins and functionally implicated in cellular processes such as axon extension, centriole stability. Fleer contains tetratricopeptide repeats (TPRs) but lacks tubulin tyrosine ligase (TTLL) motifs, a defining feature of polyglutamylase enzyme complex subunits.The C.elegans Fleer homolog DYF-1 translocates within the cilia. TPR motifs are implicated in protein interactions and we hypothesize that Fleer regulates ciliary transport of tubulin glutamylase by scaffolding an association between TTLL subunit(s) and kinesins. This proposal seeks to define the role of Fleer in biochemical process required for normal cilia formation. Specifically we will 1) assess whether Fleer exists in protein complexes together with TTLL and/or kinesin subunits and what protein subdomain(s) are required for cilia localization and protein interactions. 2) Identify novel protein interactions of Fleer using tandem affinity purification technique and yeast two hybrid assay and 3) Conditionally abrogate Fleer function in transgenic zebrafish expressing dominant negative Fleer. Successful completion of proposed studies will identify important components of the Fleer regulated network of proteins that modulate tubulin glutamylation. Components altered in the conditional knockdown model will identify key proteins involved in progression of ciliopathies that could serve as therapeutic targets. Expression analysis of different TTLL proteins in the prelimnary studies will also yield important information on how distinct tubulin modification codes are specified and provide scope for my independent career pathway. PUBLIC HEALTH RELEVANCE: Cilia and basal bodies play important roles in vertebrate development and physiology, as sensors of mechanical, chemical and osmotic stimuli. Human disease syndromes involving cystic nephropathies and sensori-neural deficits are linked to defects of cilia. Success of the proposed research will allow us to identify specific, tubulin-glutamylation dependent processes associated with ciliopathies that may underlie human disease conditions.

描述（由申请人提供）： 涉及囊性肾病和感觉神经缺陷的人类疾病综合征与纤毛缺陷有关，例如Senior-Loken综合征、Bardet-Biedl综合征、青少年肾结核。静止或运动形式的纤毛在细胞生理学中同时发挥感觉和效应功能。对衣藻和秀丽隐杆线虫等简单生物体纤毛发生的遗传和生化分析，使人们对纤毛形成所涉及的细胞过程有了详细的了解，然而，它们在纤毛疾病病理生理学中的重要性需要在脊椎动物中进行研究。在斑马鱼囊性突变体 fler 的表征中，我们发现了由于轴丝微管蛋白谷氨酰化减少而引起的广泛纤毛缺陷。多谷氨酰化是在微管蛋白上检测到的翻译后修饰，其功能涉及细胞过程，例如轴突延伸、中心粒稳定性。 Fleer 含有四肽重复序列 (TPR)，但缺乏微管蛋白酪氨酸连接酶 (TTLL) 基序，这是聚谷氨酰胺酶复合体亚基的一个决定性特征。线虫 Fleer 同源物 DYF-1 在纤毛内易位。 TPR 基序与蛋白质相互作用有关，我们假设 Fleer 通过搭建 TTLL ​​亚基和驱动蛋白之间的关联来调节微管蛋白谷氨酰胺酶的纤毛运输。该提案旨在明确 Fleer 在正常纤毛形成所需的生化过程中的作用。具体来说，我们将 1) 评估 Fleer 是否与 TTLL ​​和/或驱动蛋白亚基一起存在于蛋白质复合物中，以及纤毛定位和蛋白质相互作用需要哪些蛋白质子结构域。 2) 使用串联亲和纯化技术和酵母两种杂交测定鉴定 Fleer 的新型蛋白质相互作用，以及 3) 有条件地消除表达显性失活 Fleer 的转基因斑马鱼中的 Fleer 功能。 成功完成拟议的研究将确定弗莱尔调控的蛋白质网络的重要组成部分，这些蛋白质调节微管蛋白谷氨酰化。条件敲低模型中改变的成分将识别参与纤毛病进展的关键蛋白质，这些蛋白质可以作为治疗靶点。初步研究中不同TTLL蛋白的表达分析也将产生关于如何指定不同的微管蛋白修饰代码的重要信息，并为我的独立职业道路提供范围。 公共卫生相关性：纤毛和基体作为机械、化学和渗透刺激的传感器，在脊椎动物发育和生理学中发挥着重要作用。涉及囊性肾病和感觉神经缺陷的人类疾病综合征与纤毛缺陷有关。拟议研究的成功将使我们能够确定与纤毛病相关的特定微管蛋白谷氨酰化依赖性过程，而纤毛病可能是人类疾病的基础。