Discovery of cryptic open reading frames in Alzheimer's brain

阿尔茨海默氏症大脑中神秘开放阅读框的发现

• 批准号: 9807342
• 负责人: Chan-Hyun Na
• 金额: $ 24.56万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2021-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9807342
• 关键词: Acetylation; Alzheimer' s Disease; Amines; Brain; Brain Diseases; Cells; Codon Nucleotides; Coupled; Custom; Databases; Detection; Development; Digestion; Disease; Down-Regulation; Genes; Genetic Translation; Genome; Goals; Human; Individual; Initiator Codon; Isotope Labeling; Lead; Maps; Mass Spectrum Analysis; Messenger RNA; Methodology; Methods; Modification; N-terminal; Neurodegenerative Disorders; Nucleotides; Open Reading Frames; Pathogenesis; Peptide Initiation Factors; Peptide Mapping; Peptides; Phosphorylation; Protein Databases; Proteins; Proteome; Proteomics; Reporting; Research; Role; Sampling; Site; Technology; Translating; Translation Initiation; Translations; Up-Regulation; Work; base; differential expression; nervous system disorder; new therapeutic target; novel; ribosome profiling; subtiligase

PROJECT SUMMARY Since the beginning of -omics era, reference protein databases have defined the list of proteins are believed to be expressed in the cells. However, most proteins in the databases have been constructed by a conceptual translation of mRNAs based on a limited set of predictions rules such as that a protein translation starts at AUG that can make the longest open reading frame (ORF). Recent mass spectrometry-based proteomics and ribosome profiling studies discovered that protein translation can start at non-AUG codons, noncanonical translation initiation sites (TISs), generating small ORFs or N-terminal extension, collectively called cryptic ORFs. Strikingly, a growing number of studies have started reporting that cryptic ORFs are involved in pathogeneses of many diseases including Alzheimer’s disease (AD) that is the most prevalent neurodegenerative disease. In AD, one of the translation initiation factors, eIF2α, is inactivated by a phosphorylation on it. Consequently, eIF2A replaces the role of eIF2α switching general translation to gene- specific translation. Because eIF2A is less strict in using AUG at a TIS, when eIF2A is involved in the translation initiation, the usage of non-AUG codon is increased expressing more cryptic ORFs. Therefore, the discovery of cryptic ORFs differentially expressed in AD would have great importance in deeper understanding the AD pathogenesis mechanism. Moreover, this established method can be applicable to other neurological diseases as well. Nevertheless, the detection of those proteins has been missed due to the incomplete reference databases and technical limitations. Since ~90% of human proteins have N-terminal acetylation, this can serve as a signature modification for TISs and an in-depth identification of N-terminal acetylated peptides enable us to achieve in-depth identification of cryptic ORFs. Our group already identified over 120 cryptic ORFs from human samples using an N-terminal peptide enrichment technology, but even deeper proteome analysis for cryptic ORFs is required to cover most of the differentially expressed cryptic ORFs in AD brains. To achieve these goals, we propose three aims in this proposal. In Aim 1, we will develop a new method for the identification of N-terminal acetylated peptides by combining two different methods that were originally developed for the identification of proteolytic cleavage sites, the TAILS method and the subtiligase-based method. In Aim 2, we will determine whether acetylated peptides mapping to non-cognate codons are bona fide TISs or post-cleavage acetylated peptides using a nascent protein enrichment method. In Aim 3, we will discovery cryptic ORFs differentially expressed in AD brain using the method developed in Aim 1 as well as an in-depth total proteome analysis strategy. This project with expand and optimize our prior work on cryptic TISs, and lead to the discovery of cryptic ORFs expressed in AD brains. We hypothesize those novel cryptic ORFs are involved in the pathogenesis of AD and represent new therapeutic targets. Moreover, these approaches can be applied to the study of cryptic ORFs in other neurological diseases.

项目摘要 自组学时代开始以来，参考蛋白质数据库已经定义了被认为是 在细胞中表达。然而，数据库中的大多数蛋白质都是由概念性的 基于有限的预测规则集的mRNA翻译，例如蛋白质翻译开始于 AUG可以产生最长的开放阅读框（ORF）。最近基于质谱的蛋白质组学和 核糖体分析研究发现，蛋白质翻译可以从非AUG密码子开始， 翻译起始位点（TIS），产生小的ORF或N-末端延伸，统称为隐蔽的 ORF。引人注目的是，越来越多的研究已经开始报道，神秘的ORF参与了 包括最普遍的阿尔茨海默病（AD）在内的许多疾病的发病机制 神经退行性疾病在AD中，翻译起始因子之一eIF 2 α被 因此，eIF 2A取代了eIF 2 α的作用，将一般翻译转换为基因翻译。 具体翻译。由于eIF 2A在TIS中使用AUG时不太严格，因此当eIF 2A涉及 翻译起始时，非AUG密码子的使用增加，表达更多的隐蔽ORF。因此 发现AD中差异表达的隐藏ORF对深入了解AD具有重要意义 AD发病机制。此外，该方法还可应用于其他神经系统疾病的诊断。 疾病也是。然而，由于不完整的蛋白质检测，这些蛋白质的检测已经错过。 参考数据库和技术限制。由于~90%的人类蛋白质具有N-末端乙酰化，因此， 可作为TIS的特征修饰和N端乙酰化肽的深入鉴定 使我们能够深入鉴定隐藏的ORF。我们小组已经鉴定了120多个 使用N-末端肽富集技术从人类样本中提取ORF，但更深入的蛋白质组 需要对隐藏ORF进行分析以覆盖AD脑中大多数差异表达的隐藏ORF。 为了实现这些目标，我们在本提案中提出了三个目标。在目标1中，我们将开发一种新的方法， 通过结合两种不同的方法来鉴定N-末端乙酰化肽， 开发用于鉴定蛋白水解切割位点的TAILS方法和基于枯草杆菌酶的 法在目标2中，我们将确定映射到非同源密码子的乙酰化肽是否是正确的。 使用新生蛋白质富集方法纯化真正的TIS或裂解后乙酰化肽。在目标3中，我们 使用Aim 1中开发的方法发现AD脑中差异表达的隐藏ORF，以及 全蛋白质组分析策略。这个项目扩展和优化了我们以前在神秘TIS上的工作， 并导致发现AD脑中表达的隐蔽ORF。我们假设这些新的隐藏的ORF 参与AD的发病机制，并代表新的治疗靶点。此外，这些方法 可应用于其他神经系统疾病中隐藏ORF的研究。