Proteome-wide analysis of AD-associated SNPs

AD 相关 SNP 的全蛋白质组分析

• 批准号: 10171751
• 负责人: Heng Zhu
• 金额: $ 54.74万
• 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 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10171751
• 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，没有有效的治疗方法。全基因组关联 研究已经确定了大量的遗传变异，主要以单核苷酸的形式存在 与AD相关的多态(SNP)。阿尔茨海默病致病基因SNPs的鉴定 相关的基因变异将为疾病的病因和治疗目标提供重要的见解。 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SNPs 表情。然而，这种方法不能提供任何关于SNP结合蛋白的信息。相交 利用CHIP-SEQ技术检测带有转录因子结合位点的GWASSNPs是一种有效的鉴定功能的方法 SNPs及其相互作用的转录因子，但需要相关的转录因子的先验知识。另一种选择是确定 利用下拉耦合质谱法将差异蛋白与携带SNP的DNA片段结合； 然而，很难扩大规模以确定大量SNPs的差异结合蛋白。在这 建议，我们建议实施疾病相关单核苷酸多态蛋白质组分析(PWAS)研究 非蛋白质编码区识别等位基因特异的蛋白质-DNA/RNA相互作用和调控改变 AD中的活动。这是基于我们的假设，即功能性DNA/RNA SNP可能通过 与蛋白质的等位基因特异性相互作用。我们将研究完整的人类转铁蛋白和RNA结合蛋白 使用蛋白质阵列方法的携带SNP的DNA和RNA探针的曲目。这个化验结果也是 极高的吞吐量，因为每个探针都可以同时检测20,000个人类蛋白质。 将使用一系列生物信息学确定特定等位基因的蛋白质-DNA和-RNA相互作用的优先顺序 使用从诱导多能干细胞分化而来的人类细胞进行分析和验证。为了实现我们的目标 目标，我们提出了四个目标。目的1：确定DNA等位基因特异的SNP-Tf相互作用组。我们将表演 用凝胶移位分析验证75个AD相关SNP的蛋白质-DNA相互作用。目标2：识别RNA 等位基因特异性SNP-蛋白质相互作用组。我们将关注位于5‘-非编码区、3’-非编码区和内含子的75个SNP 可能影响RNA剪接、信使核糖核酸稳定性和蛋白质翻译的区域。目标3：评估 AD相关特定细胞类型中优先等位基因特异性相互作用的生物学后果。我们会 将蛋白质-DNA/RNA相互作用与现有的基因组数据集结合起来，以确定等位基因子集的优先顺序- 用于在人类细胞群体中验证的特定蛋白质-DNA/RNA相互作用。目标4：建设PWA 人类疾病的浏览器。我们将开发一个用户友好的PWAS浏览器来分发等位基因特定的 与人类疾病相关的蛋白质-DNA/RNA相互作用。预计这一项目的成功将 建立强大的平台并提供丰富的资源，以快速识别功能性SNP并提供关键的 对阿尔茨海默病分子机制的生物学见解。