Cellular and Molecular Features of Gene Mutations in Primary Ciliary Dyskinesia

摘要 遗传综合征原发性睫状体运动障碍(PCD)的特征是纤毛运动缺陷，导致 支气管扩张症、慢性鼻窦炎、不孕症和心脏畸形。在过去的十年里，紧张的努力已经 发现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 为了了解不同类别突变的具体细胞后果，以确定治疗途径， 类似于用于囊性纤维化的方法。纤毛的运动依赖于动力蛋白马达，动力蛋白马达固定在 纤毛骨骼轴丝上的大型复合体。编码这些动力蛋白马达的基因通常含有 PCD突变。然而，对模型生物和人类细胞的研究表明，动力蛋白必须是 由动力蛋白轴丝组装蛋白(DyAPs)在细胞质中制备。我们发现DyAP与 多纤毛细胞胞浆内的动力蛋白。DyAP的突变导致体内没有动力蛋白马达 纤毛和相应的纤毛功能受损，也导致PCD。我们最近确定了一个子集 由HEATR2/SPAG1/DNAAF2组成的DyAP，它们形成了在起始阶段发挥作用的早期支架 动力蛋白组装体。我们建议这种支架与第二组DyAP接合，我们称之为 折叠相复合体，它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平衡的细胞质聚集体 接头SQSTM1/p62，提示蛋白质加工异常导致通路中断。一致 有了这个概念，这些聚集体包含了起始阶段复合体的所有三种蛋白质。我们假设 DyAP的突变破坏了复杂的功能，导致非细胞因子的细胞内聚集体的形成 机器运转正常，动力蛋白马达未能移动到纤毛上。我们解决这个问题的目的是： (1)人类起始期DyAPs突变的功能分析，以确定它们对 与聚集体形成、细胞内转运和蛋白质与其他DyAP相互作用有关的途径 和(2)生化分析，以确定聚集体的组成和相关的活性 减少形成的细胞蛋白平衡途径。我们的目标是找出与形成 DyAP聚集体，确定DyAP途径是如何中断的，并询问聚集体的形成是否可以 被操纵以挽救足够数量的蛋白质以发挥功能，作为概念化特定的 一类PCD突变的治疗。

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.