The effect of purine stress on translation and protein stability in Leishmania

描述（由申请方提供）：利什曼原虫是一种原生动物寄生虫，可引起人类一系列疾病，对人类健康造成重大负担。目前的治疗模式完全基于少数药物，这些药物可能有毒，昂贵，难以管理，并且其有用性因耐药性的出现而受到破坏。 由于成功的寄生取决于生存的能力，即使在环境条件下，包括营养的可用性，是不利的，营养应激反应途径利什曼原虫提供了一个有吸引力的目标化疗探索。然而，很少有人知道的信号通路或分子机制，促进适应营养压力在这些寄生虫。这项研究计划的长期目标是阐明利什曼原虫的营养应激反应途径。为了探索营养应激反应，我们使用嘌呤饥饿作为范例。嘌呤收购利什曼原虫是一个必不可少的过程和饥饿的嘌呤很容易诱导文化和挑起的反应，这是强大的和听话的。我们以前的研究表明，嘌呤缺乏导致深刻的形态和代谢的变化，从寄生虫蛋白质组和代谢组的整体重组干。为了理解蛋白质组转化的分子机制，响应嘌呤饥饿，我们已经描绘了嘌呤饥饿利什曼原虫的全球转录组的变化的RNA-seq的方法。这些研究表明，在mRNA水平上观察到的变化和嘌呤饥饿细胞的蛋白质组内表现出的变化之间存在显着的不一致性，这意味着翻译和翻译后机制在嘌呤应激期间蛋白质组重构中占主导地位。这种发展性和探索性的应用的目标是建立翻译的相对贡献 以及嘌呤饥饿过程中蛋白质组动态的翻译后机制。我们将使用的方法涉及调整两种创新的代谢标记和蛋白质组学技术用于利什曼原虫，并将提供嘌呤饥饿和嘌呤充足利什曼原虫全球蛋白质合成的直接测量（目标1），以及提供蛋白质半衰期的深入研究在蛋白质组范围内（目标2）。这是第一次在利什曼原虫或其他相关病原体中使用这些技术，这些研究解决了该领域的迫切需要，提供了对翻译和翻译后稳定性的直接和定量评估，这是基因调控的两个关键领域。 在这些寄生虫身上。这些方法的成功实施也为这些和其他寄生虫的未来研究奠定了基础，这些寄生虫有望比传统上提供更广泛的翻译调控和蛋白质稳定性的研究。最后，通过将这些系统水平的结果与我们以前的蛋白质组和RNA-seq数据相结合，我们将提供关于利什曼原虫和相关病原体适应微环境应激的分子机制的最具包容性的观点之一。

DESCRIPTION (provided by applicant): Leishmania are protozoan parasites that cause a spectrum of diseases in humans and are a significant human health burden. Current treatment paradigms are based exclusively on a handful of drugs that can be toxic, costly, and difficult to administer, and whose usefulness is undermined by the emergence of drug resistance. Since successful parasitism is contingent upon an ability to subsist even when environmental conditions, including nutrient availability, are unfavorable, nutrient-stress response pathways in Leishmania offer an attractive target for chemotherapeutic exploration. However, little is known about the signaling pathways or molecular mechanisms that facilitate adaptation to nutrient-stress in these parasites. The long-term goal of this research program is to elucidate nutrient stress response pathways in Leishmania. To probe nutrient stress response we have used purine starvation as a paradigm. Purine acquisition in Leishmania is an essential process and starvation for purines is readily induced in culture and provokes a response that is both robust and tractable. Our previous studies indicate that purine scarcity leads to profound morphological and metabolic changes that stem from an overall restructuring of the parasite proteome and metabolome. To comprehend the molecular mechanisms underlying proteome transformation in response to purine starvation, we have profiled changes in the global transcriptome of purine-starved Leishmania by the method of RNA-seq. These studies indicated that a significant discordance exists between changes observed at the mRNA level and those manifested within the proteome of purine-starved cells, implying that translational and post-translational mechanisms are predominant in proteome remodeling during purine stress. The goal of this developmental and exploratory application is to establish the relative contributions of translation and post-translational mechanisms on proteome dynamics during purine starvation. The approach that we will use involves adapting two innovative metabolic labeling and proteomic technologies for use in Leishmania, and will provide a direct measure of global protein synthesis in purine-starved and purine-replete Leishmania (Aim 1), as well as affording an in-depth study of protein half-life on a proteome-wide scale (Aim 2). This is the first documented use of these techniques in Leishmania or other related pathogens, and the studies address an exigency within the field, to provide both a direct and quantitative assessment of translation and post-translational stability, two key areas of gene regulation that have defied study on a global scale in these parasites. The successful implementation of these approaches also lays the foundation for future studies in these and other parasites that promise a much broader interrogation of translational regulation and protein stability than has traditionally been afforded. Finally, by th integration of these systems-level results with our previous proteome and RNA-seq data, we will offer one of the most inclusive views of the molecular mechanisms governing adaptation to micro-environmental stress in Leishmania and related pathogens.