Identifying Protein-Protein Network Interactions between COPD Susceptibility Genes

项目摘要 复杂疾病如COPD的全基因组关联研究（GWAS）已经确定了许多基因组关联。 与疾病相关的区域，但这些遗传位点影响疾病的分子机制 发病机制在很大程度上是未知的。遗传改变的表型效应可能是由于 蛋白质之间的相互作用。然而，COPD GWAS基因座中的关键基因仅在 在少数COPD GWAS区域中得到证实，并且COPD GWAS基因之间存在网络连接， 产品很少被发现。在这个项目中，我们将利用COPD易感基因支持， GWAS和鼠肺气肿模型开发细胞类型特异性和疾病特异性蛋白质-蛋白质 与COPD相关的交互网络模块。我们假设，建立疾病网络模块的基础上， 实验验证的COPD GWAS基因之间的蛋白质-蛋白质相互作用将鉴定新的生物学活性。 影响细胞死亡、细胞衰老和/或COPD相关疾病过程的连接 炎症我们将对9种公认的 COPD GWAS基因产物。作为COPD GWAS基因之间网络链接的一部分的相互作用蛋白 基于AP-MS分析的方法将进一步用免疫共沉淀分析进行验证。我们将利用疾病 将新的AP-MS实验与现有的分子生物学实验结合起来的联合收割机网络模块构建方法 相互作用组数据，以发现COPD GWAS基因产物之间的生物学联系。我们会鉴定细胞类型- 通过基于RNA-Seq数据去除非表达基因， 支气管上皮细胞和肺泡巨噬细胞，以及疾病特异性蛋白质-蛋白质相互作用网络 基于肺组织研究联盟中来自肺组织的TOPMed RNA-Seq数据。对于网络路径 在蛋白质-蛋白质相互作用网络中连接COPD GWAS基因，我们将扰乱疾病网络模块 使用CRISPR-Cas9方法。首先，我们将评估这些扰动对蛋白质的影响， 假定疾病网络路径的相互作用。其次，我们将评估这些网络节点移除的影响 这可能导致对基于细胞的COPD相关读数的干扰，包括细胞死亡、细胞衰老和炎症。 细胞系实验中的阳性表型读数将在原代人肺细胞中得到验证。这个项目 将结合联合收割机最先进的计算和实验室方法来确定蛋白质-蛋白质相互作用 多种肺细胞类型内COPD GWAS基因之间的网络关系，并验证这些网络 使用基于细胞的相关生物学过程的读数来确定COPD发病机制的关系。

PROJECT SUMMARY Genome-wide association studies (GWAS) of complex diseases like COPD have identified many genomic regions associated with disease, but the molecular mechanisms by which these genetic loci influence disease pathogenesis are largely unknown. The phenotypic effects of genetic alterations can result from disruptions of the coordinated interactions between proteins. However, the key genes within COPD GWAS loci have only been proven in a minority of those COPD GWAS regions, and network connections between the COPD GWAS gene products have rarely been identified. In this project, we will utilize COPD susceptibility genes supported by both GWAS and murine emphysema models to develop cell type-specific and disease-specific protein-protein interaction network modules related to COPD. We hypothesize that building disease network modules based on experimentally validated protein-protein interactions between COPD GWAS genes will identify novel biological connections that influence COPD-related disease processes of cell death, cellular senescence, and/or inflammation. We will perform affinity purification/mass spectrometry (AP-MS) assays of nine well-established COPD GWAS gene products. Interacting proteins that are part of network links between COPD GWAS genes based on the AP-MS assays will be further validated with co-immunoprecipitation assays. We will utilize disease network module building approaches that combine the new AP-MS experiments with existing molecular interactome data to find biological linkages between COPD GWAS gene products. We will identify cell type- specific protein-protein interaction networks by removing non-expressed genes based on RNA-Seq data in bronchial epithelial cells and alveolar macrophages, and disease-specific protein-protein interaction networks based on TOPMed RNA-Seq data from lung tissue in the Lung Tissue Research Consortium. For network paths linking COPD GWAS genes in the protein-protein interaction network, we will perturb disease network module components using CRISPR-Cas9 approaches. First, we will assess the effects of these perturbations on protein interactions of putative disease network paths. Second, we will assess the impact of these network node removal perturbations on cell-based COPD-related readouts, including cell death, cellular senescence, and inflammation. Positive phenotypic readouts in cell line experiments will be validated in primary human lung cells. This project will combine state-of-the-art computational and laboratory approaches to identify the protein-protein interaction network relationships between COPD GWAS genes within multiple lung cell types and to validate those network relationships using cell-based readouts of relevant biological processes for COPD pathogenesis.