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

描述（由申请人提供）：利什曼原虫是引起人类一系列疾病的原生动物寄生虫，是人类重大的健康负担。目前的治疗模式完全基于少数几种药物，这些药物可能有毒、昂贵、难以施用，而且耐药性的出现削弱了这些药物的有效性。由于成功的寄生依赖于即使在环境条件（包括养分供应）不利的情况下生存的能力，利什曼原虫的营养应激反应途径为化疗探索提供了一个有吸引力的目标。然而，对于促进这些寄生虫适应营养胁迫的信号通路或分子机制知之甚少。本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。在利什曼原虫中嘌呤的获取是一个重要的过程，嘌呤的缺乏在培养中很容易被诱导，并引发一种既强大又可处理的反应。我们之前的研究表明，嘌呤的缺乏会导致寄生虫蛋白质组和代谢组的全面重组导致深刻的形态和代谢变化。为了了解嘌呤缺乏时蛋白质组转化的分子机制，我们通过RNA-seq方法分析了嘌呤缺乏利什曼原虫的全球转录组变化。这些研究表明，mRNA水平的变化与嘌呤缺乏细胞蛋白质组内的变化存在显著不一致，这意味着嘌呤胁迫下蛋白质组重塑的主要机制是翻译和翻译后机制。这种发展和探索性应用的目的是确定翻译的相对贡献

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.