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

描述（由申请人提供）：从包括人类在内的广泛不同物种的基因组编码的短开放阅读框（sorf）中直接翻译的少于100个氨基酸的肽，最近被发现具有重要的生物学功能，包括通过人类肽humanin预防疾病相关的细胞凋亡。这些基因组编码肽（GEPs）不仅挑战了生物活性肽生产的规范蛋白水解模型，而且挑战了我们对基因构成的理解。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，需要对这些肽进行完整的目录。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GEPs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，选择性地从细胞肽丘富集GEPs，允许检测和发现低丰度的GEPs。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不太可能被核糖体扫描翻译。因此，我们将研究这些gep的翻译起始机制，并具体确定它们是否由内部核糖体进入位点的内部起始产生。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础的新的生物学见解，而且还将提供具有潜在生物医学相关性的新一类基因产物的完整目录。

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. PUBLIC HEALTH RELEVANCE: The proposed research aims to identify and rigorously characterize bioactive peptides encoded directly in the human genome and to investigate the mechanism by which they are translated. Literature precedent demonstrates that genomically encoded peptides (GEPs) have important (patho)physiologial activities; one example is the human GEP humanin, which protects neurons against disease-related apoptosis. The proposed research, in expanding the number of known GEPs, will reveal many more peptides of this class that have physiological functions in human cells and potential roles in disease progression or prevention.