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

摘要 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起作用。发现于小核糖核酸病毒中 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。虽然经常被称为“自我 “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 核糖体。因此，我们发现 2A 肽在贾第鞭毛虫中效果不佳，这表明其在 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。我们假设 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，我们将检验这个假设，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。我们会 将遗传、生化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，目标是寻找新的有效且功能性的 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 描述真核生物中肽键跳跃的机制。总体而言，这项工作将提供关键 关于贾第鞭毛虫核糖体功能的知识，真核生物翻译的基本工作原理 机制，以及2A肽功能的机制。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。

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.