The role of outer dynein arm docking complex components in primary ciliary dyskinesia

原发性纤毛运动障碍（PCD）是一种常染色体隐性遗传病，由纤毛运动缺陷引起，以上呼吸道和下呼吸道感染为特征。外动力蛋白臂（oda）是附着在外微管双链A上的多蛋白复合物，产生纤毛和鞭毛跳动的动力。外动力蛋白臂对接复合体（ODA-DC）为外动力蛋白臂提供对接位点，因此缺乏ODA-DC会导致轴突体的oda缺乏。2013年，我们在CCDC114突变患者的呼吸细胞轴突组中鉴定出ARMC4为ODA-DC相关蛋白。在此之后，我们确定了额外的ODA-DC成分，如CCDC151， TTC25和MNS1，并表征了它们在PCD中的功能作用。最近，我们发现了一种新的与人类ODA-DC相关的候选Ca2+结合蛋白（EFCAB1），我们目前正在对其进行表征。根据我们的数据，ODA-DC似乎在不同物种之间存在差异，而且在呼吸纤毛和结纤毛以及精子鞭毛之间也存在差异。需要对不同类型的细胞进行更深入的分析，以阐明这种复杂功能在人体中的结构和机制。关于ODA-DC组成的额外知识对于改进PCD诊断程序和患者护理非常重要。因此，本提案的目标如下：参与ODA对接的新型钙结合蛋白EFCAB1和参与ODA 2型组装的DAW1的分子和细胞特性研究由于ODA-DC功能异常导致PCD的其他基因缺陷的鉴定候选蛋白在对照和突变组织中的表达分析验证在模式生物涡虫中的功能作用研究5。ODA- dc相关蛋白与ODA蛋白相互作用研究用原代人气道培养物的蛋白质组学分析鉴定ODA-DC缺陷多蛋白ODA-DC复合物的蔗糖梯度分馏表征。遵循这一策略，我们期望我们将1)成功检测与ODA-DC相关的新成分，2)成功表征人类纤毛中ODA-DC的组成和细胞类型特异性差异的更详细特征。这些数据将提高我们对正常纤毛生物学的理解，并将为PCD的发病机制提供更多的见解，从而改善受影响个体的诊断和遗传咨询。此外，我们希望我们将能够开发新的治疗策略PCD。

Primary ciliary dyskinesia (PCD) is an autosomal recessive disorder resulting from defective ciliary motility and characterized by upper and lower respiratory infections. Outer dynein arms (ODAs) are multiprotein complexes attached to the outer microtubule doublet A generating the motive force for ciliary and flagellar beating. The outer dynein arm docking complex (ODA-DC) provides the docking sites for the outer dynein arm so that an absence of ODA-DC results in a deficiency in the ODAs from the axonemes. In 2013, we identified ARMC4 as an ODA-DC associated protein undetectable in the axonemes of respiratory cells from patients with CCDC114 mutations. Following this, we identified additional ODA-DC components such as CCDC151, TTC25 and MNS1 and characterized their functional role in PCD. Recently, we identified a new candidate Ca2+-binding protein to be associated with ODA-DC in human (EFCAB1) and we are currently working on its characterization. Based on our data, ODA-DC appears to differ between the different species but also between the respiratory and nodal cilia as well as in sperm flagella. A deeper analysis of the different cell types is needed to elucidate the structure and the mechanism by which this complex function in human. Additional knowledge about the composition of the ODA-DC is of great importance to improve PCD diagnostic procedures and patient care. The objectives of this current proposal are therefore as follows: 1. Molecular and cellular characterization of the novel calcium-binding protein EFCAB1 involved in ODA docking and DAW1 involved in ODA type 2 assembly in human PCD individuals 2. Identification of additional gene defects resulting in PCD due to abnormal ODA-DC function 3. Validation of candidate proteins by expression analyses in control and mutant tissue 4. Investigation of the functional role in the model organism planaria 5. Protein interaction studies of ODA-DC related and ODA proteins 6. Characterization of the ODA-DC defects by proteomic analysis of primary human airway cultures 7. Characterization of the multiprotein ODA-DC complex using sucrose gradient fractionation. Following this strategy, we expect that we will 1) succeed to detect novel components associated with the ODA-DC and 2) succeed to characterize the composition of the ODA-DC in human cilia and cell type specific differences in more detail. These data will improve our understanding of normal cilia biology and which will give additional insights into the pathogenesis of PCD, thus improving diagnosis and genetic counseling of affected individuals. In addition, we hope that we will be able to develop novel therapeutic strategies for PCD.