Discovery of cryptic open reading frames in Alzheimer's brain

项目总结 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通过概念性的 基于一组有限的预测规则(例如蛋白质翻译开始于 8月，可以形成最长的开放阅读框(ORF)。最新基于质谱学的蛋白质组学和 核糖体图谱研究发现，蛋白质翻译可以从非AUG密码子开始，非规范的 翻译起始点(TI)，产生小的ORF或N末端延伸，统称为秘密 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 许多疾病的发病机制，包括最常见的阿尔茨海默病(AD) 神经退行性疾病。在AD中，翻译起始因子之一eif2α被一种 它被磷酸化了。因此，eIF2a取代了eif2α的角色，将一般翻译转换为基因- 具体的翻译。因为eIF2A在TIS中使用Aug时不那么严格，所以当eIF2A参与 翻译启动时，非Aug密码子的使用增加了，表达了更多神秘的ORF。因此， 发现阿尔茨海默病差异表达的神秘开放阅读框对于深入理解AD具有重要意义 AD的发病机制。此外，该方法也适用于其他神经科。 疾病也是如此。然而，由于不完全的原因，这些蛋白质的检测被遗漏了。 参考数据库和技术限制。由于~90%的人类蛋白质有N-末端乙酰化，这 可作为TISS的签名修饰和N-末端乙酰化肽的深入鉴定 使我们能够实现对神秘ORF的深入识别。我们的小组已经发现了120多个神秘的 使用N端肽浓缩技术从人类样本中提取ORF，但蛋白质组更深 需要对隐蔽ORF进行分析，以涵盖AD大脑中大多数差异表达的隐蔽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来 两种不同方法相结合鉴定N-末端乙酰化肽 开发用于鉴定蛋白水解性裂解位点、尾巴方法和基于枯草杆菌酶的 方法。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BonA 使用新生蛋白富集法的真实TISS或裂解后乙酰化肽。在《目标3》中，我们将 使用目标1中开发的方法发现AD脑中差异表达的隐蔽ORF以及 深入的总蛋白质组分析策略。这个项目扩展和优化了我们之前在神秘TIL上的工作， 并导致发现了在AD大脑中表达的神秘的ORF。我们假设那些新奇神秘的ORF 参与了AD的发病机制，代表了新的治疗靶点。此外，这些方法 可应用于其他神经系统疾病中隐匿ORF的研究。

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.