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

描述(申请人提供)：最近发现，由编码在包括人类在内的广泛不同物种的基因组中编码的短开放阅读框(SORF)直接翻译的少于100个氨基酸的多肽具有重要的生物学功能，包括通过人多肽Human in防止与疾病相关的细胞凋亡。这些基因组编码的多肽(GEP)不仅挑战了生物活性多肽生产的典型蛋白分解模型，而且挑战了我们对基因组成的理解。然而，目前只有少数人的GEP被报道，因此在我们了解它们的全部生物活性之前，需要一个这些肽的完整目录。因此，我们将应用基于液质联用(LC-MS)的多肽组学方法在人类细胞系中大规模发现GEP。随后的有针对性的基因敲除和过度表达研究将被用于试验性地将新的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多肽中富含GEP，从而能够检测和发现低丰度的GEP。最后，我们的初步结果揭示了GEP编码在注释RNA深处的ORF中，这不太可能通过核糖体扫描来翻译。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多肽的产生和翻译启动机制提供基本的新的生物学见解，而且还将提供一类具有潜在生物医学意义的新基因产品的完整目录。

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.