Small-molecule signals controlling nematode development

项目摘要/摘要 线虫秀丽线虫依靠小分子信号控制其发育、代谢、 这些信号在许多寄生线虫物种中发挥着保守的作用。这 米拉应用概述了我们正在进行的努力，以了解结构，生物合成和机制 几类重要的小分子信号，包括(1)蛔虫苷--一大类 线虫分泌的信息素，蠕虫用来诱导抗应激的达尔幼虫阶段和 协调各种行为，(2)N-乙酰谷氨酰胺NACQ#1--雄性优先分泌的信息素 为了对抗达尔诱导的蛔虫苷对两性人的影响，促进生殖发育， 和(3)线酰胺-一族杂合的聚酮-非核糖体肽，在体内充当激素 通过一种鲜为人知的机制来蠕虫和促进饥饿生存。我们的目标是生物合成的 通过在蠕虫基因组中产生精确突变来在体内实现这些信号分子的途径 CRISPR-Cas9，并分析这些突变对猪的初级和次级代谢组的影响 蠕虫使用比较代谢组学。这种方法使我们能够将生物合成途径映射到这些 天然产物，以确定由这些途径产生的额外信号分子，并确定如何 这些途径与蠕虫中的其他代谢途径相交。我们严格确认具体的作用 途径中的酶通过使用体外酶分析重建途径，有机合成 生物合成中间体和结构研究。在这项裁决的支持下，我们将调查 线虫酰胺的生物合成和机制以及两种必需的和神秘的生物作用 产生线胺的神经元是神经管相关神经元(CAN)。我们将使用线虫胺 生物合成，这需要分布在整个蠕虫基因组中的基因，以了解 复杂次生代谢物的生物合成是在动物系统的背景下控制的。此外， 我们将研究这种生物合成途径如何与其他次生生物合成途径相交。 代谢物，包括蛔虫苷和NACQ#1。一个中心焦点将是蠕虫如何调节 这些不同的小分子信号的产生和运输响应不同的因素和 以协调其发育、新陈代谢和生理的环境条件。这项工作将 深入了解线虫和其他线虫物种如何利用小分子信号控制重要的 保守下游信号通路，如胰岛素途径。此外，考虑到守恒性 对于寄生线虫物种中的这些小分子信号，这项工作将提供新的化学工具和 干扰这些线虫生命周期的策略。

Project Summary/Abstract The nematode Caenorhabditis elegans relies on small-molecule signals to control its development, metabolism, physiology, and behavior, and these signals play conserved roles in many parasitic nematode species. This MIRA application outlines our ongoing efforts to understand the structures, biosynthesis, and mechanisms of several important classes of small-molecule signals, including (1) the ascarosides – a broad family of pheromones secreted by C. elegans that the worm uses to induce the stress-resistant dauer larval stage and to coordinate various behaviors, (2) the N-acyl glutamine nacq#1 – a pheromone that males preferentially secrete to counter the effects of dauer-inducing ascarosides on hermaphrodites and promote reproductive development, and (3) the nemamides – a family of hybrid polyketide-nonribosomal peptides that serve as hormones in the worm and promote starvation survival through a poorly understood mechanism. We target the biosynthetic pathways to these signaling molecules in vivo by generating precise mutations in the worm genome using CRISPR-Cas9 and analyzing the effects of these mutations on the primary and secondary metabolome of the worm using comparative metabolomics. This approach enables us to map the biosynthetic pathways to these natural products, to identify additional signaling molecules produced by these pathways, and to determine how these pathways intersect with other metabolic pathways in the worm. We rigorously confirm the role of specific enzymes in the pathways by reconstituting the pathways using in vitro enzymatic assays, organic synthesis of biosynthetic intermediates, and structural studies. With the support of this award, we will investigate the biosynthesis and mechanism of the nemamides, as well as the biological roles of the two essential and enigmatic neurons where the nemamides are produced, the canal-associated neurons (CANs). We will use nemamide biosynthesis, which requires genes that are distributed throughout the worm genome, to understand how the biosynthesis of complex secondary metabolites is controlled in the context of an animal system. Furthermore, we will investigate how this biosynthetic pathway intersects with the biosynthetic pathways of other secondary metabolites, including the ascarosides and nacq#1. A central focus will be how the worm regulates the production and trafficking of these different small-molecule signals in response to different factors and environmental conditions in order to coordinate its development, metabolism, and physiology. This work will provide insights into how C. elegans and other nematode species use small-molecule signals to control important conserved downstream signaling pathways, such as the insulin pathway. Furthermore, given the conservation of these small-molecule signals in parasitic nematode species, this work will provide new chemical tools and strategies to interfere with the life cycles of those nematodes.