Small-molecule signals controlling nematode development

项目总结/摘要 秀丽隐杆线虫依靠小分子信号来控制其发育，代谢， 生理和行为，这些信号在许多寄生线虫物种中发挥保守的作用。这 MIRA应用程序概述了我们正在进行的努力，以了解结构，生物合成，和机制， 小分子信号的几个重要类别，包括（1）Ascarosides -一个广泛的家族， C.线虫用来诱导抗应激的幼虫期， 协调各种行为，（2）N-酰基谷氨酰胺nacq#1 -雄性优先分泌的信息素 为了对抗雌虫诱导的子囊苷对雌雄同体的影响并促进生殖发育， 和（3）线虫酰胺--一个杂合聚酮-非核糖体肽家族，其在哺乳动物中充当激素。 蠕虫和促进饥饿生存通过一个知之甚少的机制。我们的目标是生物合成 通过在蠕虫基因组中产生精确的突变， CRISPR-Cas9突变并分析这些突变对肿瘤细胞的初级和次级代谢组的影响。 使用比较代谢组学。这种方法使我们能够绘制这些生物合成途径 天然产物，以确定这些途径产生的其他信号分子，并确定如何 这些途径与蠕虫中的其他代谢途径交叉。我们严格确认特定的 通过使用体外酶测定法重建途径， 生物合成中间体和结构研究。在这个奖项的支持下，我们将调查 生物合成和机制的线虫，以及生物学作用的两个重要和神秘的 神经元中产生的神经酰胺，运河相关神经元（CAN）。我们将使用nemamide 生物合成，这需要基因分布在整个蠕虫基因组，以了解如何 复杂次级代谢物的生物合成在动物系统中受到控制。此外，委员会认为， 我们将研究这种生物合成途径如何与其他次生代谢物的生物合成途径交叉， 代谢物，包括子囊苷和nacq#1。一个中心焦点将是蠕虫如何调节 产生和运输这些不同的小分子信号，以响应不同的因素， 环境条件，以协调其发展，代谢和生理。这项工作将 深入了解C.线虫和其他线虫物种使用小分子信号来控制重要的 保守的下游信号通路，如胰岛素通路。此外，考虑到保护 这些小分子信号在寄生线虫物种，这项工作将提供新的化学工具， 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.