IDENTIFICATION OF PTMS ON RIBOKINASE FROM LEISHMANIA MEXICANA

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 利什曼原虫是一种单细胞原生动物，在许多发展中国家造成毁灭性的疾病，针对这种疾病的药物治疗有限。对于这些寄生虫来说，葡萄糖是一种核心重要的营养物质，这些寄生虫是由促进葡萄糖转运蛋白家族的许可输入的。兰达杰实验室通过有针对性的基因替换在墨西哥乳杆菌中产生了一个葡萄糖转运蛋白缺失突变体，该缺失突变体具有强大的生物学表型，包括无法在巨噬细胞内生存和复制，巨噬细胞是被寄生虫入侵的哺乳动物宿主细胞。通过对野生型和突变型寄生虫的双向凝胶电泳和质谱分析，已经鉴定了40多种蛋白质，它们的水平在这两个品系之间发生了显著变化。其中一种这样的蛋白质，核激酶(RK)，在缺失突变体中上调了~7倍，可能是为了通过另一条途径补充核糖-磷酸，在运输缺陷的突变体中，核糖-磷酸不能有效地从葡萄糖中合成。2D凝胶的Western blotting显示，在零突变体中出现了4个新的RK斑点，其分子量与野生型RK相似。这些结果表明，葡萄糖饥饿诱导了RK的特异性翻译后修饰(PTM)。为了开始研究导致这些修饰的信号转导途径，我们建议与PNNL合作，鉴定葡萄糖饥饿诱导的RK上的PTM。最初，将在RK上进行完整蛋白的LC-MS以识别特定的PTM。随后，对RK多肽进行自下而上的分析，以确定RK上的特定修饰位点。虽然利什曼原虫在其生命周期中必须对环境的各种变化做出反应，包括葡萄糖供应的显著变化，但在分子水平上几乎没有人知道它们如何适应这种环境变化。这个重点项目将开始详细绘制这些病原体因葡萄糖限制而产生的生化修饰。该项目将为最终组装一条生化途径奠定基础，揭示这些寄生虫如何适应营养水平的变化，从而在它们的生命周期中维持宿主的生存能力。

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 parasites are single cell protozoa that cause devastating diseases throughout much of the developing world and for which limited pharmacological therapies exist. Glucose is a nutrient of central importance for these parasites that is imported by permeases of the facilitative glucose transporter family. The Landfear laboratory has generated a glucose transporter null mutant in L. mexicana by targeted gene replacement, and this null mutant has strong biological phenotypes including the inability to survive and replicate within macrophages, the mammalian host cell that is invaded by the parasite. Analysis of wild type and null mutant parasites by 2-dimensional (2D) gel electrophoresis and mass spectrometry (MS) has identified over 40 proteins whose levels change significantly between these two lines. One such protein, ribokinase (RK), is upregulated ~7-fold in the null mutants, probably to replenish by an alternative route ribose-phosphate that cannot be made efficiently from glucose in the transport-deficient mutant. Western blots of 2D gels revealed that 4 new RK spots, of similar MW to wild type RK, appear in the null mutant. These results suggest that glucose starvation induces specific post-translational modifications (PTMs) on RK. To begin to examine the signal transduction pathway that results in these modifications, we propose to partner with PNNL to identify the PTMs that are induced on RK by glucose starvation. Initially, intact protein LC-MS will be performed on RK to identify the specific PTMs. Subsequently bottom-up analysis of RK peptides will be employed to identify the specific sites of modifications on RK. Although Leishmania parasites must respond to a variety of changes in environment during their life cycles, including pronounced changes in glucose availability, almost nothing is known at the molecular level about how they adapt to such environmental alterations. This focused project will begin to map in detail biochemical modifications in these pathogens that ensue from glucose limitation. The project will form the basis for ultimately assembling a biochemical pathway revealing how these parasites adapt to altering nutrient levels and thus maintain viability within their hosts during their life cycle.