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

基本信息

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
项目类别：
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

项目摘要

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纤毛运动障碍 (PCD) 的特点是纤毛运动缺陷，导致支气管扩张、慢性鼻窦炎、不孕症和心脏畸形。经过近十年的不懈努力发现了纤毛组装所需的途径，并发现了 PCD 中的突变基因。下一个目标是了解不同类别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骨骼轴丝上的大复合体。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，动力蛋白必须是由动力蛋白轴丝组装蛋白 (DyAP) 在细胞质中制备。我们发现 DyAP 与多纤毛细胞胞质灶中的动力蛋白。 DyAP 突变导致体内动力蛋白马达缺失纤毛，因此纤毛功能受损，也会导致 PCD。我们最近确定了一个子集由 HEATR2/SPAG1/DNAAF2 组成的 DyAP，形成早期支架，在起始阶段发挥作用动力蛋白组装。我们建议该支架与第二组 DyAP 结合，我们将其称为折叠相复合体，进行动力蛋白组装以运输至纤毛。我们观察到突变在起始阶段，DyAP 导致形成带有蛋白质稳态标记的细胞质聚集体衔接子 SQSTM1/p62，表明异常的蛋白质加工导致通路中断。持续的根据这个概念，这些聚集体包含起始相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-聚集体的形成机械功能正常，动力蛋白马达无法移动到纤毛。我们回答这个问题的目的如下： (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、细胞内运输以及与其他 DyAP 的蛋白质相互作用相关的途径 (2) 生化分析，以确定聚集体的组成和相关活性细胞蛋白质稳态途径以减轻形成。我们的目标是确定与形成相关的因素 DyAP 聚集，确定 DyAP 途径如何被中断，并询问是否可以形成聚集体操纵以拯救足够量的蛋白质以发挥功能，作为概念化特定的第一步针对一类 PCD 突变的治疗。