Scope and mechanism of eptide translation from short open reading frames in the h

短开放阅读框肽翻译的范围和机制

• 批准号: 8314323
• 负责人: Sarah Ann Slavoff
• 金额: $ 5.22万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-01 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8314323
• 关键词: Amino Acid Sequence; Amino Acids; Apoptosis; Azides; Binding; Biochemistry; Biological; Biological Phenomena; Biological Process; Biology; Cataloging; Catalogs; Cell physiology; Cells; Chemicals; Data Set; Detection; Disease; Disease Progression; Elements; Genes; Genetic; Genome; Human; Human Cell Line; Human Genome; In Vitro; Internal Ribosome Entry Site; Investigation; K-562; Literature; Messenger RNA; Methionine; Modeling; Mutation; Neurons; Open Reading Frames; Peptides; Physiological; Post-Translational Protein Processing; Production; Protein Precursors; Proteins; Proteolysis; Proteome; RNA Sequences; Relative (related person); Reporting; Research; Ribosomes; Role; Scanning; Shotguns; Small Interfering RNA; Stress; Structure; Transcript; Translating; Translation Initiation; Translations; Validation; Viral; analog; base; disorder prevention; humanin; insight; knock-down; liquid chromatography mass spectrometry; overexpression; protein aminoacid sequence; synthetic peptide; tool; translation assay

DESCRIPTION (provided by applicant): Peptides of fewer than 100 amino acids that are directly translated from short open reading frames (sORFs) encoded in the genomes of widely divergent species, including human, have recently been discovered to have important biological functions, including prevention of disease-related apoptosis by the human peptide humanin. These genomically encoded peptides (GEPs) challenge not only the canonical proteolytic model of bioactive peptide production but also our understanding of what constitutes a gene. However, only a handful of human GEPs have been reported, so a complete catalog of these peptides is required before we can understand the full scope of their bioactivities. We will therefore apply a liquid chromatography-mass spectrometry (LC-MS)-based peptidomics approach to large-scale GEP discovery in human cell lines. Subsequent targeted genetic knock-downs and overexpression studies will be used to experimentally assign new GEPs to the short genes that encode them. We will also use the tools of chemical biology to selectively enrich GEPs from the cellular peptidome, permitting detection and discovery of low-abundance GEPs. Finally, our preliminary results reveal GEPs encoded in ORFs deep inside annotated RNAs, which are unlikely to be translated by ribosome scanning. We will therefore investigate the mechanism of translation initiation for these GEPs, and specifically determine if they are produced by internal initiation at internal ribosome entry sites. This project will not only provide fundamental new biological insights into mechanisms of bioactive peptide production and translation initiation, but will also provide a complete catalog of a new class of gene products with potential biomedical relevance.

描述（由申请人提供）：最近发现，从广泛不同物种（包括人）的基因组中编码的短开放阅读框（sORF）直接翻译的少于100个氨基酸的肽具有重要的生物学功能，包括通过人肽humanin预防疾病相关的细胞凋亡。这些基因组编码的肽（GEP）不仅挑战了生物活性肽生产的经典蛋白水解模型，也挑战了我们对基因组成的理解。然而，只有少数人类GEP被报道，因此在我们了解其生物活性的全部范围之前，需要这些肽的完整目录。因此，我们将应用基于液相色谱-质谱（LC-MS）的肽组学方法在人类细胞系中大规模发现GEP。随后的靶向基因敲除和过表达研究将用于实验性地将新的GEP分配给编码它们的短基因。我们还将使用化学生物学的工具来选择性地从细胞肽组中富集GEP，从而允许检测和发现低丰度的GEP。最后，我们的初步结果揭示了GEPs编码在注释RNA深处的ORF中，这些ORF不太可能通过核糖体扫描进行翻译。因此，我们将研究这些GEP的翻译起始机制，并特别确定它们是否是由内部核糖体进入位点的内部起始产生的。该项目不仅将为生物活性肽的产生和翻译起始机制提供基本的新生物学见解，还将提供具有潜在生物医学相关性的一类新基因产物的完整目录。