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

总结 蓝氏贾第鞭毛虫是一种单细胞真核生物，每年感染数亿人。因为贾第虫 与其他真核生物相比，它具有许多简化的分子途径，因此它具有作为 非传统模式系统，用于研究关键生物过程的多样性和进化。 我们最近偶然发现，2A“自切割”肽序列在细胞中的作用非常差。 贾第虫，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， 它们能够从一个开放阅读框产生两个多肽。虽然经常被称为“自我”， 切割，2A肽通过使核糖体跳过一个肽键来起作用。其机制是 未知的，但必须涉及2A新生肽链和出口通道之间的特异性相互作用。 核糖体因此，我们发现2A肽在贾第虫中作用不佳，这表明其在贾第虫中的基本差异。 核糖体相比，其他真核生物，并可以利用了解2A行动的机制。 对我们最近解决的贾第虫80 S核糖体结构的检查揭示了一个引人注目的差异 在出口通道中的核糖体蛋白uL 4的结构中：贾第虫在uL 4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 解释为什么2A肽在贾第虫体内的作用很差。在这里，我们将测试这个假设，并在这样做（1）定义 2A肽诱导键跳跃的机制和（2）确定它在贾第虫中失败的原因。我们将 联合收割机将遗传、生物化学和结构方法结合在两个目标中。在第一个目标中，我们将确定 2A序列变体在贾第虫中发挥作用的程度，目的是找到新的有效和功能性的 这些序列将成为贾第虫研究人员的有力工具。在第二个目标中，我们将直接测试 uL 4环的功能作用，并通过cryo-EM解决T2 A-核糖体复合物的结构，目标是 描述了真核生物中肽键跳跃的机制。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，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虫的发展 作为一种模式生物，并帮助奠定基础，为新的抗病毒治疗，阻断2A肽的活性。

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.