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: 7532925
• 负责人: Narendra H Pathak
• 金额: $ 13.66万
• 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/7532925
• 关键词: Adolescent; Affinity Chromatography; Antibodies; Axon; Bardet-Biedl Syndrome; Biochemical; Biochemical Genetics; Biochemical Process; Biological Assay; C-terminal; Caenorhabditis elegans; Cell physiology; Cells; Centrioles; Chemicals; Chlamydomonas; Cilia; Co-Immunoprecipitations; Code; Complex; Condition; Coupled; Defect; Development; Disease; Dominant-Negative Mutation; Embryo; Enzymes; Epitopes; Family member; Functional disorder; Genes; 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; Public Health; 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; intracellular protein transport; kinetosome; mutant; novel; protein localization location; 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、青少年肾病综合征。纤毛以其静止或运动的形式在细胞生理学中起着感觉和效应器的作用。通过对衣原体和秀丽隐杆线虫等简单生物体纤毛发生的遗传和生物化学分析，我们对纤毛形成的细胞过程有了深入的了解，但它们在纤毛疾病病理生理学中的重要性还需要在脊椎动物中进行研究。在表征斑马鱼囊性突变fleer，我们确定了广泛的纤毛缺陷所产生的轴丝微管蛋白的谷氨酰化减少。多聚谷氨酰化是微管蛋白上检测到的翻译后修饰，并且在功能上涉及细胞过程，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物亚基的一个定义特征。秀丽隐杆线虫Fleer同源物DYF-1在纤毛内移位。TPR基序与蛋白质相互作用有关，我们假设Fleer通过构建TTLL亚基与驱动蛋白之间的关联来调节微管蛋白谷氨酰胺酶的纤毛转运。该建议旨在确定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亚结构域。2)使用串联亲和纯化技术和酵母双杂交试验鉴定Fleer的新型蛋白质相互作用; 3）有条件地消除表达显性负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调节微管蛋白谷氨酰化的Fleer调节蛋白质网络的重要组成部分。在条件性敲除模型中改变的组分将鉴定参与纤毛病变进展的关键蛋白质，其可以作为治疗靶点。在初步研究中对不同TTLL蛋白的表达分析也将产生关于如何指定不同微管蛋白修饰代码的重要信息，并为我的独立职业生涯提供范围。 公共卫生相关性：纤毛和基体作为机械、化学和渗透刺激的感受器，在脊椎动物的发育和生理中起着重要作用。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特定的，微管蛋白-谷氨酰化依赖的过程与纤毛病变，可能是人类疾病的基础。