Proteome-wide analysis of AD-associated SNPs

AD 相关 SNP 的全蛋白质组分析

基本信息

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
项目类别：
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&apos Untranslated Regions 5&apos Untranslated Regions Affect Affinity Alleles Alzheimer&apos 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

项目摘要

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.

项目概要阿尔茨海默病（AD）是一种神经退行性疾病，目前尚无有效治愈方法。全基因组关联研究（GWAS）已经鉴定出大量的遗传变异，大部分以单核苷酸的形式存在与 AD 相关的多态性 (SNP)。 AD 中致病 SNP 的鉴定相关的遗传变异将为该疾病的病因学和治疗靶点提供重要的见解。表达数量性状位点 (eQTL) 研究可以识别可能影响下游基因的 SNP 表达。然而，这种方法无法提供有关 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 是识别功能性的有用方法 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，很难扩大规模来鉴定大量 SNP 的差异结合蛋白。在这个提案中，我们建议对疾病相关 SNP 进行全蛋白质组分析 (PWAS) 研究非蛋白质编码区，用于识别等位基因特异性蛋白质-DNA/RNA 相互作用和调节的改变 AD 中的活动。这是基于我们的假设，即功能性 DNA/RNA SNP 可能通过以下方式执行其功能：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，包含携带 SNP 的 DNA 和 RNA 探针。该测定也是通量极高，因为每个探针可以同时检测 >20,000 个人类蛋白质。将使用一系列生物信息学对已识别的等位基因特异性蛋白质-DNA 和-RNA 相互作用进行优先排序使用诱导多能干细胞分化的人类细胞进行分析和验证。为了实现我们的目标，我们提出四个目标。目标 1：确定 DNA 等位基因特异性 SNP-TF 相互作用组。我们将表演通过凝胶迁移分析验证了 75 个 AD 相关 SNP 的蛋白质-DNA 相互作用分析。目标 2：识别 RNA 等位基因特异性 SNP 蛋白相互作用组。我们将重点关注位于 5'-UTR、3'-UTR 和内含子的 75 个 SNP 这些区域可能会影响 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中进行验证的特定蛋白质-DNA/RNA 相互作用。目标 4：构建 PWAS 人类疾病浏览器。我们将开发一个用户友好的 PWAS 浏览器来分发特定等位基因与人类疾病相关的蛋白质-DNA/RNA 相互作用。预计该项目的成功建立强大的平台，提供丰富的资源，快速识别功能性SNP，提供关键的SNP AD 分子机制的生物学见解。