Cellular and Molecular Features of Gene Mutations in Primary Ciliary Dyskinesia

原发性纤毛运动障碍基因突变的细胞和分子特征

• 批准号: 10378548
• 负责人: Steven Brody
• 金额: $ 56.85万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10378548
• 关键词: Address; Animal Model; Appearance; Autophagocytosis; Behavior; Biochemical; Biological Assay; Bronchiectasis; Cells; Characteristics; Chronic; Chronic Sinusitis; Chronic lung disease; Cilia; Code; Complex; Consequentialism; Cystic Fibrosis; Cytoplasm; Data; Defect; Disease; Dynein ATPase; Failure; Fluorescence Resonance Energy Transfer; Gene Mutation; Genes; Genetic; Genetic Diseases; Genetic study; Goals; Heart Abnormalities; Heat shock factor; Human; Image; Impairment; Infertility; Inherited; Interruption; Lead; Lung diseases; Mass Spectrum Analysis; Missense Mutation; Modeling; Molecular; Molecular Chaperones; Motor; Mutate; Mutation; Nature; Neurodegenerative Disorders; Pathologic; Pathway interactions; Phase; Primary Ciliary Dyskinesias; Process; Proteins; Regulatory Pathway; Structural Protein; Structure; Syndrome; Therapeutic; Transmission Electron Microscopy; candidate marker; cell motility; chronic respiratory disease; cilium biogenesis; cilium motility; gene discovery; member; motility disorder; motor impairment; multicatalytic endopeptidase complex; mutant; protein aggregation; protein degradation; protein function; protein protein interaction; proteostasis; recurrent infection; scaffold; skeletal; trafficking

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.

概括 遗传综合征原发性睫状运动障碍（PCD）的特征是纤毛运动的缺陷，导致 支气管扩张，慢性鼻窦炎，不育和心脏畸形。过去十年来 纤毛组装所需的未覆盖途径，并发现了PCD中​​突变体的基因。下一个目标是 了解不同类别突变的特定细胞后果以识别治疗途径， 类似于用于囊性纤维化的方法。纤毛运动取决于固定在 纤毛的骨骼轴突上的大型复合物。这些为这些动力蛋白电动机编码的基因通常是港口 PCD突变。但是，模型生物和人类细胞的研究表明，动力蛋白必须是 通过动力蛋白轴突组装蛋白（Dyaps）在细胞质中制备。我们发现与 多细胞细胞的细胞质灶中的动力蛋白。染料中的突变导致在内部没有动力蛋白电动机 纤毛和纤毛功能受损，也导致PCD。我们最近确定了一个子集 由Heatr2/spag1/dnaaf2组成的染料，该染料在起始阶段形成了早期脚手架 Dynein组装。我们建议这种脚手架与第二组染料互动，我们称 折叠相的复合物，该复合物将动力蛋白组件运输到纤毛。我们观察到突变 在开始期间，染料会导致形成具有蛋白质的标记的细胞质聚集体 适配器SQSTM1/P62，表明异常蛋白质加工会导致途径中断。持续的 有了这个概念，这些聚集体包含起始阶段复合物的所有三种蛋白质。我们假设 染色体中的突变中断复合功能，导致形成非 - 的细胞内聚集体 运行机械和未能将动力蛋白电动机移至纤毛。我们以这些目的解决了这个问题： （1）对引发相染料的人类突变的功能分析，以确定其对 与聚集体形成，细胞内运输和蛋白质相互作用有关的途径 （2）生化分析，以确定聚集体的组成和相关的活性 细胞蛋白抑制途径减轻形成。我们的目标是确定与形成有关的因素 DYAP聚集体，确定Dyap途径如何中断，并询问骨料形成是否可以是 操纵以挽救足够数量的蛋白质以进行功能，作为概念化特定的第一步 一类PCD突变的处理。