Proteome-wide analysis of AD-associated SNPs

AD 相关 SNP 的全蛋白质组分析

• 批准号: 10405083
• 负责人: Heng Zhu
• 金额: $ 53.98万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-30 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10405083
• 关键词: 3' Untranslated Regions; 5' Untranslated Regions; Affect; Affinity; Alleles; Alzheimer' s Disease; Base Pairing; Binding; Binding Proteins; Binding Sites; Bioinformatics; Biological; Biological Assay; Cell Differentiation process; Cell physiology; Cells; Coupled; DNA; DNA Probes; DNA-Protein Interaction; Databases; Disease; EMSA; Effectiveness; Electrophoretic Mobility Shift Assay; Etiology; Gene Expression; Genes; Genetic; Genetic Risk; Genetic Transcription; Genetic study; Genomics; Goals; Human; Immunoprecipitation; In Vitro; Individual; Introns; Investigation; Kinetics; Knock-in; Knowledge; Lead; Length; Location; Luciferases; Maps; Mass Spectrum Analysis; Measures; Molecular; Names; Neurodegenerative Disorders; Neurons; Outcome; Population; Protein Array; Protein Microchips; Proteins; Proteome; Quantitative Trait Loci; RNA; RNA Probes; RNA Splicing; RNA Stability; RNA Transport; RNA-Binding Proteins; RNA-Protein Interaction; Regulatory Element; Resources; Series; Single Nucleotide Polymorphism; Specificity; Surveys; Technology; Testing; Translations; Untranslated RNA; Untranslated Regions; Validation; age related neurodegeneration; base; cell type; chromatin immunoprecipitation; genetic risk factor; genetic variant; genome wide association study; genomic data; high throughput screening; human disease; induced pluripotent stem cell; insight; knock-down; mRNA Stability; molecular sequence database; novel; overexpression; predictive test; risk variant; scale up; screening; success; therapeutic target; transcription factor; transcriptome sequencing; user-friendly

Project Summary Alzheimer's disease (AD) is a neurodegenerative disorder with no effective cure. Genome-wide association studies (GWAS) have identified a large number of genetic variants, mostly in the form of single nucleotide polymorphisms (SNPs), that are associated with AD. Identification of the causative SNPs among the AD- associated genetic variants will provide important insights into etiology of the disease and therapeutic targets. Expression quantitative trait loci (eQTLs) studies can identify SNPs that are likely to affect downstream gene expression. However, this approach cannot provide any information on SNP-binding proteins. Intersecting GWAS SNPs with transcription factor (TF) binding sites by ChIP-seq is a useful approach to identify functional SNPs and their interacting TFs but requires a priori knowledge of the relevant TFs. Another option is to identify differential protein binding to a SNP-carrying DNA fragment using pull down-coupled mass spectrometry; however, it is difficult to scale up to identify differential binding proteins for a large number of SNPs. In this proposal, we propose to implement a Proteome-Wide Analysis of disease-associated SNPs (PWAS) study on non-protein coding regions to identify allele-specific protein-DNA/RNA interactions and alteration of regulatory activity in AD. This is based on our hypothesis that functional DNA/RNA SNPs likely execute their function via allele-specific interactions with proteins. We will survey the entire human TF and RNA-binding protein repertoires with SNP-carrying DNA and RNA probes using a protein array-based approach. This assay is also extremely high-throughput because >20,000 human proteins can be simultaneously surveyed for each probe. Identified allele-specific protein-DNA and -RNA interactions will be prioritized using a series of bioinformatics analyses and validated using human cells differentiated from induced pluripotent stem cells. To achieve our goals, we propose four aims. Aim 1: Determine DNA allele-specific SNP-TF interactome. We will perform protein-DNA interaction assay with 75 AD-associated SNPs validated by gel-shift assays. Aim 2: Identify RNA allele-specific SNP-protein interactome. We will focus on 75 SNPs located in 5'-UTR, 3'-UTR and intronic regions, which are likely to affect RNA splicing, mRNA stability, and protein translation. Aim 3: Assess the biological consequences of prioritized allele-specific interactions in AD relevant specific cell types. We will integrate the protein-DNA/RNA interactions with existing genomic datasets to prioritize a subset of allele- specific protein-DNA/RNA interactions for validation in human cell populations. Aim 4: Construct a PWAS browser for human diseases. We will develop a user-friendly PWAS browser to distribute the allele-specific protein-DNA/RNA interactions that are relevant to human diseases. The success of this project is expected to establish a powerful platform and provide a rich resource to rapidly identify functional SNPs and provide crucial biological insights into the molecular mechanisms underlying AD.

项目摘要 阿尔茨海默病（AD）是一种神经退行性疾病，目前尚无有效的治疗方法。全基因组关联 研究（GWAS）已经确定了大量的遗传变异，大多数是单核苷酸的形式， 多态性（SNP）与AD相关。在AD-10中鉴定致病性SNP。 相关的遗传变异将为疾病的病因学和治疗靶点提供重要的见解。 表达数量性状基因座（eQTL）研究可以识别可能影响下游基因的SNP， 表情然而，这种方法不能提供关于SNP结合蛋白的任何信息。相交 通过ChIP-seq检测具有转录因子（TF）结合位点的GWAS SNP是一种有用的方法， SNP及其相互作用的TF，但需要相关TF的先验知识。另一种选择是识别 使用下拉偶联质谱法与携带SNP的DNA片段的差异蛋白质结合; 然而，难以按比例放大以鉴定大量SNP的差异结合蛋白。在这 根据这项建议，我们建议实施一项疾病相关SNP的蛋白质组分析（PWAS）研究， 非蛋白质编码区，以鉴定等位基因特异性蛋白质-DNA/RNA相互作用和调节基因的改变。 活动AD。这是基于我们的假设，即功能性DNA/RNA SNP可能通过以下途径执行其功能： 与蛋白质的等位基因特异性相互作用。我们将调查整个人类TF和RNA结合蛋白 使用基于蛋白质阵列的方法，用携带SNP的DNA和RNA探针检测基因组库。该分析也是 非常高的通量，因为每个探针可以同时检测> 20，000种人类蛋白质。 确定等位基因特异性蛋白质-DNA和-RNA相互作用将优先使用一系列的生物信息学 使用从诱导多能干细胞分化的人细胞进行分析和验证。实现我们 我们提出了四个目标。目的1：确定DNA等位基因特异性SNP-TF相互作用组。我们将执行 蛋白质-DNA相互作用试验，75个AD相关SNP通过凝胶迁移试验验证。目标2：识别RNA 等位基因特异性SNP-蛋白质相互作用组。我们将重点研究位于5 '-UTR、3'-UTR和内含子中的75个SNPs 这些区域可能影响RNA剪接、mRNA稳定性和蛋白质翻译。目标3：评估 AD相关特定细胞类型中优先的等位基因特异性相互作用的生物学后果。我们将 将蛋白质-DNA/RNA相互作用与现有的基因组数据集整合，以优先考虑等位基因的子集， 用于在人类细胞群体中验证的特异性蛋白质-DNA/RNA相互作用。目标4：构建PWAS 人类疾病的浏览器。我们将开发一个用户友好的PWAS浏览器， 与人类疾病相关的蛋白质-DNA/RNA相互作用。预计该项目的成功将 建立了一个强大平台，提供了丰富的资源，以快速识别功能SNPs，并提供关键的 对AD潜在分子机制的生物学见解。