The Role of Dynein Motor Mutations in Motile Cilia Disease

项目摘要 运动纤毛对肺防御是必不可少的，这一点可以从遗传综合征原发性纤毛运动障碍中得到证实 （PCD）。PCD的特征是运动纤毛受损，导致出生时呼吸窘迫，随后是慢性 鼻窦炎和支气管扩张，这可能导致呼吸衰竭。没有特定的治疗方法 对于PCD，部分原因是运动纤毛生物发生和发病机制的关键途径尚未确定。PCD具有 与近40个基因的突变有关。那些编码动力蛋白的蛋白质， 跳动，包括最大类的PCD突变。我们发现动力蛋白马达蛋白的突变 导致纤毛细胞中异常的细胞质聚集。重要的是，这些聚集体螯合正常的 组装动力蛋白运动复合体所需的机器蛋白，这表明全球性的破坏， 纤毛组装为了进一步确定突变的影响，我们对患者的气道细胞进行了活检和培养。 与PCD，揭示增加的表达与细胞应激相关的基因，包括IL-1 β和IL 6。我们假设 突变蛋白质的积累导致纤毛组装机制的失败和细胞应激。这 将通过以下具体目标进行测试：（1）确定突变动力蛋白蛋白如何中断 纤毛运动装配途径和（2）定义细胞中的转录和应激反应， PCD突变蛋白。我们将利用原代培养鼻细胞从患者的突变动力蛋白运动 在我们的PCD诊所发现的蛋白质。为了确定突变蛋白如何干扰纤毛装配，我们将使用蛋白质组学 以及先进的显微镜来量化突变动力蛋白与组装机器的相互作用。 为了表征突变蛋白对细胞应激的影响，我们将采用RNA测序来确定突变蛋白的结构。 PCD细胞的转录谱和测试已知的细胞应激途径。本提案生成的数据将 识别PCD中的共享途径，可用于开发未来的治疗策略。 该提案包括一项计划，为Horani博士提供指导研究，技术技能发展， 和量身定制的教学培训需要实现他的目标，成为一个独立的物理学家，科学家。的 培训将涵盖遗传学和基因组学、测序数据分析和高级荧光 显微成像，这是该提案的关键领域。这个项目将由一个科学顾问监督 该委员会拥有运动纤毛生物学、蛋白质相互作用、蛋白质抑制途径和成像方面的专业知识。的 委员会将确保实现职业里程碑，完成正式课程工作，并与 在当地和国际上都有发展。通过拟议的研究产生的结果可以应用于 其他遗传性气道疾病和培训将使Horani博士能够开发新的方法和疗法， 作为一名独立的研究者，可以改善患者的健康状况。

PROJECT SUMMARY Motile cilia are essential for lung defense, as evidenced by the genetic syndrome primary ciliary dyskinesia (PCD). PCD is characterized by impaired motile cilia resulting in respiratory distress at birth, followed by chronic sinopulmonary infection and bronchiectasis, which can lead to respiratory failure. There are no specific therapies for PCD, in part because key pathways for motile cilia biogenesis and pathogenesis are not defined. PCD has been linked to mutations in nearly 40 genes. Those that encode dynein proteins, the motors necessary for cilia beating, comprise the largest class of PCD mutations. We have found that mutations in dynein motor proteins result in abnormal cytoplasmic aggregates in ciliated cells. Importantly, these aggregates sequester normal proteins of the machinery required to assemble the dynein motor complexes, suggesting a global disruption of cilia assembly. To further determine the impact of mutations, we biopsied and cultured airway cells from patients with PCD, revealing increased expression of genes related to cell stress, including IL-1 and IL6. We hypothesize that accumulation of mutant protein leads to failure of the cilia assembly machinery and to cellular stress. This will be tested through the following Specific Aims: (1) Determine how mutant dynein proteins interrupt the cilia motor assembly pathway and (2) Define the transcriptional and stress responses in cells containing PCD mutant proteins. We will leverage primary culture nasal cells from patients with mutations in dynein motor proteins seen at our PCD clinic. To determine how mutant proteins perturb cilia assembly, we will use proteomics and advanced microscopy to quantify the interaction of the mutant dynein proteins with the assembly machinery. To characterize the effect of mutant protein on cell stress, we will employ RNA sequencing to define the transcriptional profile of PCD cells and test known cell stress pathways. Data generated from this proposal will identify shared pathways in PCD, that can be exploited to develop future therapeutic strategies. This proposal comprises a plan to provide Dr. Horani with the mentored research, technical skill development, and tailored didactic training needed to achieve his goal of becoming an independent physician-scientist. The training will cover areas of genetics and genomics, sequencing data analysis, and advanced fluorescent microscopy imaging, which are key areas of this proposal. This project will be overseen by a scientific advisory committee with expertise in motile cilia biology, protein interactions, proteostatic pathways and imaging. The committee will ensure that career milestones are realized, formal course work is completed, and collaborations are developed locally and internationally. Findings generated through the proposed studies can be applied to other genetic airway disease and training will allow Dr. Horani to develop new approaches and therapies that may improve patients’ health as an independent investigator.