Self-cleaving peptides: Mechanisms and Use in Diverse Eukaryotic Species

自裂解肽：机制及其在不同真核物种中的应用

基本信息

批准号：
10678481
负责人：
Olivia Selfridge Rissland
金额：
$ 19.14万
依托单位：
UNIVERSITY OF COLORADO DENVER
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-09-01 至 2025-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10678481
关键词：
Amino Acid Sequence Antiviral Agents Biochemical Biological Biological Assay Biological Models Biological Process Biology Biomedical Research Cells Complex Cryoelectron Microscopy Development Elements Eukaryota Event Evolution Family Picornaviridae Foot-and-Mouth Disease Virus Foundations Future Generations Genetic Genome engineering Giardia Giardia lamblia Giardiasis Goals Growth Harvest Human Human poliovirus Intestinal Diseases Knowledge Life Cycle Stages Mammals Medicine Modeling Molecular Open Reading Frames Organism Oryctolagus cuniculus Parasites Pathway interactions Peptides Peptidyltransferase Persons Positioning Attribute RNA Recording of previous events Reporter Research Research Personnel Reticulocytes Ribosomal Proteins Ribosomes Role Saccharomyces cerevisiae Structural Models Structure System Technology Testing Therapeutic Time Translations Variant Viral Virus Diseases Work experimental study human pathogen interest model organism novel novel therapeutic intervention polypeptide protein aminoacid sequence stem tool

项目摘要

SUMMARY Giardia lamblia is a single-cell eukaryote that infects hundreds of millions of people every year. Because Giardia has many molecular pathways that are simplified compared to other eukaryotes, it has potential as a nontraditional model system for studying the diversity and evolution of key biological processes. We recently serendipitously discovered that the 2A ‘self-cleaving’ peptide sequences work very poorly in Giardia, surprising because 2A peptides are thought to work universally in eukaryotes. Found in picornaviruses like foot-and-mouth disease virus and poliovirus, 2A peptides are an essential part of the viral life cycle because they enable two polypeptides to be produced from one open reading frame. Although often referred to as ‘self- cleaving,’ 2A peptides operate by causing the ribosome to skip a peptide bond. The mechanism of this is unknown but must involve specific interactions between the 2A nascent peptide chain and the exit tunnel of the ribosome. Thus, our discovery that 2A peptides work poorly in Giardia points at fundamental differences in its ribosomes compared to other eukaryotes and can be exploited to understand the mechanism of 2A action. Examination of our recently solved structure of the Giardia 80S ribosome reveals a compelling difference in the structure of ribosome protein uL4 in the exit channel: Giardia lacks a specific loop in uL4. We hypothesize that this loop is important for the peptide bond-skipping mechanism of 2A peptides, and its absences can partially explain why 2A peptides operate poorly in Giardia. Here, we will test this hypothesis and in so doing (1) define the mechanism by which 2A peptides induce bond skipping and (2) determine why it fails in Giardia. We will combine genetic, biochemical, and structural approaches in two aims. In the first aim, we will determine the extent to which 2A sequence variants can function in Giardia, with the goal of finding novel efficient and functional sequences that will serve as powerful tools for Giardia researchers. In the second aim, we will directly test the functional role of the uL4 loop and solve the structure of a T2A-ribosome complex by cryo-EM with the goal of describing the mechanism of peptide bond skipping in eukaryotes. Overall, this work will provide critical knowledge about the function of the Giardia ribosome, the fundamental workings of the eukaryotic translational machinery, and the mechanism of 2A peptide function. Our discoveries will facilitate the development of Giardia as a model organism and help lay the foundation for new anti-viral therapeutics that block 2A peptide activity.

概括蓝氏贾第鞭毛虫是一种单细胞真核生物，每年感染数亿人，因为贾第鞭毛虫。与其他真核生物相比，它具有许多简化的分子途径，因此具有作为用于研究关键生物过程的多样性和进化的非传统模型系统。我们最近偶然发现 2A“自切割”肽序列在贾第鞭毛虫，令人惊讶，因为 2A 肽被认为在真核生物中普遍存在，在小核糖核酸病毒中也有发现。与口蹄疫病毒和脊髓灰质炎病毒一样，2A 肽是病毒生命周期的重要组成部分，因为它们能够从一个开放阅读框产生两种多肽，尽管通常被称为“自体”。 2A 肽通过使核糖体跳过肽键来发挥作用。其机制是。未知，但必须涉及 2A 新生肽链和 2A 出口通道之间的特定相互作用因此，我们发现 2A 肽在贾第鞭毛虫中效果不佳，这表明其存在根本差异。核糖体与其他真核生物相比，可用于了解 2A 作用机制。对我们最近解决的贾第鞭毛虫 80S 核糖体结构的检查揭示了一个引人注目的差异退出通道中核糖体蛋白 uL4 的结构：贾第鞭毛虫在 uL4 中缺乏特定的环。这个环对于 2A 肽的肽键跳跃机制很重要，它的缺失可以部分地影响解释为什么 2A 肽在贾第鞭毛虫中效果不佳。在这里，我们将检验这一假设，并在此过程中 (1) 定义。 2A 肽诱导键跳跃的机制，以及 (2) 确定它在贾第鞭毛虫中失败的原因。将遗传、生物化学和结构方法结合起来实现两个目标。在第一个目标中，我们将确定 2A序列变体在贾第鞭毛虫中发挥作用的程度，目的是寻找新的有效变体和功能序列将成为贾第鞭毛虫研究人员的强大工具在第二个目标中，我们将直接测试 uL4 环的功能作用并通过冷冻电镜解析 T2A-核糖体复合物的结构，目标是总的来说，这项工作将提供关键的描述真核生物中肽键跳跃的机制。关于贾第鞭毛虫核糖体功能的知识，真核翻译的基本工作原理我们的发现将促进贾第鞭毛虫的发展。作为一种模式生物，有助于为阻断 2A 肽活性的新抗病毒疗法奠定基础。