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。因为贾迪亚 与其他真核生物相比，它具有许多简化的分子途径，具有作为 用于研究关键生物过程多样性和进化的非传统模型系统。 我们最近偶然发现，2a‘自裂解’的多肽序列在 贾第虫，令人惊讶的是，因为2A肽被认为在真核生物中普遍起作用。在小核糖核酸病毒中发现 与口蹄疫病毒和脊髓灰质炎病毒一样，2A多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中产生两个多肽。尽管经常被称为“自我” 在切割过程中，‘2a肽通过使核糖体跳过一个肽键来运作。这其中的机制是 未知，但必须涉及新生的2A多肽链和 核糖体。因此，我们发现，在贾第虫中，2A多肽的作用很差，这是因为它在其 核糖体与其他真核生物相比，可以被用来理解2A的作用机制。 对我们最近解决的贾第虫80S核糖体结构的检查发现了一个令人信服的差异 在出口通道中核糖体蛋白uL4的结构中：贾第虫在uL4中缺乏一个特定的环。我们假设 这个环对于2A肽的肽键跳跃机制是重要的，它的缺失可以部分地 解释为什么2A肽在贾第虫体内的作用不佳。在这里，我们将检验这一假设，并在此过程中定义(1) 2A多肽诱导跳键的机制和(2)决定其在贾第虫中失败的原因。我们会 在两个目标中结合遗传、生化和结构方法。在第一个目标中，我们将确定 在贾第鞭毛虫中，为了找到新的高效和功能性的目标，2A序列变体可以在多大程度上发挥作用 这将成为贾第虫研究人员的强大工具。第二个目标，我们将直接测试 UL4环的功能作用和用冷冻EM解决T2A-核糖体复合体的结构，目的是 描述真核生物中的肽键跳跃机制。总的来说，这项工作将提供关键的 了解贾第虫核糖体的功能，即真核细胞翻译的基本工作原理 机制，以及2A多肽的作用机制。我们的发现将促进贾第虫的发展 作为一种模式生物，并有助于为阻断2A肽活性的新的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.