COMPARATIVE PROTEOMICS TO DEFINE ADAPTATIONS TO PURINE STRESS IN LEISHMANIA

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 利什曼原虫是原生动物寄生虫，在人类中引起显著的发病率和死亡率，困扰着全世界约1200万人。这些寄生虫是嘌呤营养缺陷型的，并且已经进化出一套独特的嘌呤转运蛋白和补救酶，以从其宿主中吸收这些必需的营养素。因此，嘌呤补救组分为药物设计提供了有吸引力的靶标。适应嘌呤环境变化的能力对于利什曼原虫在宿主中生存至关重要。细胞外嘌呤的撤回导致关键嘌呤转运蛋白和补救酶的急性增加。这种增加不是由于mRNA丰度或稳定性的增加。由于调控是转录后的，因此我们的目标是使用蛋白质组学方法来深入了解这种嘌呤应激适应中所涉及的细胞网络和途径。提出了两个战略。首先，定量嘌呤饥饿后杜氏利什曼原虫蛋白质组的时间变化，一个准确的质量和时间（AMT）标签策略划定。一个广泛的AMT数据库将构建从无嘌呤和嘌呤充满条件下生长的寄生虫，并用于促进高通量准确测量嘌呤饥饿和嘌呤充满寄生虫之间的相对蛋白质丰度。其次，由于嘌呤应激反应的一部分涉及调节蛋白的翻译后修饰，因此嘌呤饥饿和嘌呤充足的L.多诺万将被比较。预计这些研究将产生广泛的影响，阐明利什曼原虫适应宿主嘌呤环境变化的方法，并可能为未来的治疗探索提供新的靶点和途径。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Leishmania are protozoan parasites that cause significant morbidity and mortality in humans, afflicting approximately 12 million people worldwide. These parasites are auxotrophic for purines and have evolved a unique set of purine transporters and salvage enzymes to scavenge these essential nutrients from their host. Hence, purine salvage components provide attractive targets for drug design. An ability to adapt to changes in the purine milieu is vital for Leishmania to survive in their host. Withdrawal of extracellular purines leads to an acute increase in key purine transporters and salvage enzymes. This augmentation is not due to an increase in mRNA abundance or stability. Since regulation is post-transcriptional, it is our goal to use proteomic methods to provide insight into the cellular networks and pathways involved in this adaptation to purine stress. Two strategies are proposed. First, to quantify the temporal changes in the Leishmania donovani proteome upon purine-starvation, an accurate mass and time (AMT) tag strategy is delineated. A broad AMT database will be constructed from parasites grown under purine-free and purine-replete conditions and used to facilitate high-throughput accurate measurements on relative protein abundance between purine-starved and purine-replete parasites. Second, since it is conjectured that part of the response to purine stress involves the post-translational modification of regulatory proteins, the phosphoproteome of purine-starved and purine-replete L. donovani will be compared. It is anticipated that these studies will be of broad impact, illuminating the means by which Leishmania adapt to changes in their host purine environment and likely providing novel targets and pathways for future therapeutic exploration.