Cellular and Molecular Features of Gene Mutations in Primary Ciliary Dyskinesia

总结 遗传综合征原发性纤毛运动障碍（PCD）的特征是纤毛运动缺陷，导致 支气管扩张、慢性鼻窦炎、不孕症和心脏畸形。在过去十年中， 揭示了纤毛组装所需的途径，并发现了PCD中的突变基因。下一个目标是 为了了解不同类型突变的特定细胞后果以确定治疗途径， 类似于用于囊性纤维化的方法。纤毛运动依赖于动力蛋白马达，动力蛋白马达固定在纤毛中。 纤毛骨架轴丝上的大复合体。编码这些动力蛋白马达的基因通常含有 PCD突变。然而，对模式生物和人类细胞的研究表明，动力蛋白必须是 在细胞质中由动力蛋白轴丝组装蛋白（DyAP）制备。我们发现DyAP与 多纤毛细胞胞质灶中的动力蛋白。DyAP的突变导致细胞内动力蛋白马达的缺失， 纤毛和因此受损的纤毛功能，也导致PCD。我们最近发现了 由HEATR 2/SPAG 1/DNAAF 2组成的DyAP，其形成早期支架以在起始阶段发挥功能 动力蛋白的组装我们提出，这种支架与第二组DyAP结合，我们称之为 折叠相复合物，其进行动力蛋白组装以运输到纤毛。我们观察到突变 在起始阶段，DyAP导致形成标记有蛋白质抑制的细胞质聚集体 衔接子SQSTM 1/p62，表明异常的蛋白质加工导致通路中断。一致 根据这一概念，这些聚集体含有起始阶段复合物的所有三种蛋白质。我们假设 DyAP的突变中断了复杂的功能，导致细胞内非- 机械功能和动力蛋白马达无法移动到纤毛。我们处理这一问题的目的是： (1)对起始阶段DyAP的人类突变进行功能分析，以确定其对 与聚集体形成、细胞内运输和蛋白质与其他DyAP相互作用相关的途径 和（2）生物化学分析，以鉴定聚集体的组成和聚集体的相关活性。 细胞蛋白质代谢抑制途径以减轻形成。我们的目标是确定与形成 DyAP聚集体，确定DyAP通路是如何中断的，并询问聚集体的形成是否可以被阻断。 操纵以拯救足够数量的蛋白质用于功能，作为概念化特定蛋白质的第一步。 治疗一类PCD突变。

Summary The genetic syndrome primary ciliary dyskinesia (PCD) is characterized by defects in cilia motility resulting in bronchiectasis, chronic sinusitis, infertility and cardiac malformations. Intense efforts over the past decade have uncovered pathways required for cilia assembly and discovered genes that are mutant in PCD. The next goal is to understand the specific cellular consequences of different classes of mutations to identify therapeutic avenues, similar to the approach used for cystic fibrosis. Cilia motility is dependent on dynein motors, which are fixed in large complexes on the skeletal axoneme of cilia. Genes that code for these dynein motors commonly harbor PCD mutations. However, studies in model organisms and human cells have revealed that dyneins must be prepared in the cytoplasm by dynein axoneme assembly proteins (DyAPs). We found that DyAPs colocalize with dyneins in cytoplasmic foci of multiciliated cells. Mutation in DyAPs results in an absence of dynein motors within the cilia and consequentially, impaired cilia function, also resulting in PCD. We have recently identified a subset of DyAPs composed of HEATR2/SPAG1/DNAAF2, which form an early scaffold to function in an initiation phase of dynein assembly. We propose that this scaffold engages with a second group of DyAPs, that we term the folding phase complex, which carries out dynein assembly for transport to the cilia. We observed that mutations in the initiation phase DyAPs result in the formation of cytoplasmic aggregates tagged with the proteostasis adapter SQSTM1/p62, suggesting that abnormal protein processing leads to pathway interruption. Consistent with this concept, these aggregates contain all three proteins of the initiation phase complex. We hypothesize that mutations in DyAPs interrupt the complex function, lead to the formation of intracellular aggregates of non- functioning machinery and a failure to move dynein motors to the cilia. We address this question with these aims: (1) A functional analysis of human mutations of the initiation phase DyAPs to determine their effect on the pathway related to the formation of aggregates, intracellular trafficking, and protein interactions with other DyAPs and (2) a biochemical analysis to identify the composition of the aggregates and the associated activity of the cellular proteostasis pathways to mitigate formation. Our goal is to identify factors related to the formation of DyAP aggregates, determine how the DyAP pathway is interrupted and ask if aggregates formation can be manipulated to rescue sufficient amounts of protein for function, as a first step toward conceptualizing a specific treatment for one class of PCD mutations.