Fleer: Functional study in regulation of vertebrate cilia tublin modification

描述(由申请人提供)： 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，High-Loken综合征、Bardet-Biedl综合征、青少年肾炎。在细胞生理学中，纤毛以其静止或移动的形式既具有感觉功能又具有效应器功能。对衣藻和线虫等简单生物纤毛发生的遗传和生化分析使人们对纤毛形成的细胞过程有了详细的了解，但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。在斑马鱼囊状突变体Fleer的特征中，我们发现了广泛的纤毛缺陷，这些缺陷是由于轴丝微管蛋白谷氨酰化减少引起的。多聚谷氨酰化是在微管蛋白上检测到的一种翻译后修饰，在功能上与细胞过程有关，如轴突延伸、中心粒稳定。线虫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，这是聚谷氨酸酶复合体亚基的一个特征。线虫Fleer同源基因DYF-1易位在纤毛内。TPR基序与蛋白质之间的相互作用有关，我们假设Fleer通过构建Ttll亚单位(S)和动蛋白之间的联系来调节微管蛋白谷氨酸酶的纤毛运输。这项建议试图确定Fleer在正常纤毛形成所需的生化过程中的作用。具体地说，我们将1)评估Fleer是否与TTL1和/或Kinesin亚基一起存在于蛋白质复合体中，以及纤毛定位和蛋白质相互作用需要哪些蛋白质亚域(S)。2)利用串联亲和纯化技术和酵母双杂交技术鉴定Fleer新的蛋白质相互作用；3)在表达显性负性Fleer的转基因斑马鱼中条件性地取消Fleer功能。 成功完成拟议的研究将确定Fleer调控的调节微管蛋白谷氨酰化的蛋白质网络的重要组成部分。条件性基因敲除模型中改变的成分将识别参与纤毛疾病进展的关键蛋白质，这些蛋白质可以作为治疗靶点。在初步研究中，对不同TTLL蛋白的表达分析也将为如何指定不同的微管蛋白修饰密码提供重要信息，并为我的独立职业生涯提供范围。 与公共健康相关：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扮演着重要的角色。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疾病相关的特定的、微管-谷氨酰胺依赖的过程，这些过程可能是人类疾病的基础。

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.