The development of tools for proteomic analysis in trypanosomes

描述（由申请人提供）：该项目的目标是开发高通量蛋白质组学工具，以加强对锥虫的研究。锥虫是一种原生动物寄生虫，在发展中国家造成重大的公共卫生和经济问题。三种不同锥虫的基因组序列已经完成，因此下一步是对这些生物体中所有蛋白质的表征。亲和纯化和质谱是常规用于表征蛋白质的强大工具，并且已经成为蛋白质组学研究中不可或缺的工具。然而，这两种方法都需要适当的样品制备才能产生高质量的结果，而且真核生物基因组编码的蛋白质数量庞大，这意味着需要高通量方法来快速系统地分析单个蛋白质产生的所有可能的相互作用。由于这些原因，我们正在调整和开发用于酵母研究的技术用于锥虫的研究，例如：(i)冰解，一种裂解冷冻细胞以保存蛋白质复合物的方法，因为它们在收集时；（ii） 96孔高通量筛选，使用最少的细胞物质，快速简便地确定任何蛋白质复合物的最佳缓冲条件；（iii）随机或靶向相互作用的同位素区分（I-DIRT）——一种使用稳定同位素标记区分特异性和非特异性相互作用蛋白质的成熟方法。为了在锥虫中开发和验证这些方法，我们选择使用核孔复合物（npc）的一些组成蛋白（称为Nups）作为测试平台。NPCs是细胞核和细胞质之间唯一的交换介质；每个NPC是一个约50MDa的大分子组装，由30个不同的nup组成，总共约480个拷贝。我们选择NPC是因为它代表了各种各样的蛋白质-蛋白质相互作用类型（因为它参与核运输，核糖核蛋白复合物组装，细胞周期控制和染色质修饰复合物），也因为我们之前在布鲁氏锥虫（TbNups）中鉴定和gfp标记了22个Nups，这样所有的Nups现在都携带了一个方便的亲和处理。我们将首先使用所有已识别的TbNups优化方法。然后我们将重点关注TbNup92，这是一种在有丝分裂期间重新定位到纺锤体组织者的Nup。纺锤体蛋白质组的成功定义将证明我们的蛋白质组学方法可以访问高度动态的细胞周期调节过程。接下来，我们将扩展这些蛋白质组学工具来探索层；细胞核内与核膜密切相关并参与细胞核结构和功能的调节，如染色质组织和基因转录的细丝网最后，我们将在其他锥虫细胞器的选择目标上测试这些方法，以确认它们广泛适用，并确保可转移到合作实验室。有了基因组序列和蛋白质数据库提供的丰富信息，我们相信我们寻求开发的方法将不仅对锥虫的蛋白质组学研究有益，而且最终对其他寄生原生生物有益。

DESCRIPTION (provided by applicant): The objective of this project is to develop high throughput proteomic tools to enhance the study of trypanosomes - protozoan parasites that cause major public health and economic problems across the developing world. The genomic sequence of three different trypanosomes has been completed, so the next step is the characterization of all the proteins in these organisms. Affinity purification and mass spectrometry are powerful tools that are routinely used to characterize proteins, and have become virtually indispensable for proteomics research. However, both methods require appropriate sample preparation to yield quality results, and the sheer number of proteins that are encoded by eukaryotic genomes means that high throughput methods are required to rapidly and systematically analyze all possible interactions made by individual proteins. For these reasons, we are adapting and developing techniques used in the study of yeast to trypanosome research, such as: (i) cryolysis, a method for lysing frozen cells in order to preserve protein complexes as they were at the time of collection; (ii) a 96-well high throughput screen to determine optimal buffer conditions for any protein complex in a fast and facile procedure using minimal amount of cellular material; (iii) Isotopic Differentiation of Interactions as Random or Targeted (I-DIRT) - a proven method for distinguishing between specifically and nonspecifically interacting proteins using stable isotopic labeling. To develop and validate these methods in trypanosomes, we have chosen to use as a test bed a select number of component proteins (termed Nups) of nuclear pore complexes (NPCs). NPCs are the sole mediators of exchange between the nucleus and the cytoplasm; each NPC is a ~50MDa macromolecular assembly composed of 30 different Nups present in a total of ~480 copies. We chose the NPC because it represents a wide variety of protein-protein interaction types (as it is involved in nuclear transport, ribonucleoprotein complex assembly, cell cycle control and chromatin modifying complexes) and also because we previously identified and GFP-tagged 22 Nups in Trypanosoma brucei (TbNups), such that all now carry a convenient affinity handle. We will first optimize the methods using all identified TbNups. We will then focus on TbNup92, a Nup that relocates to the spindle organizer during mitosis. Successful definition of a spindle proteome will demonstrate our proteomic approach can access highly dynamic, cell cycle regulated processes. Next, we will expand these proteomic tools to explore the lamina; a meshwork of filaments within the nucleus that are intimately associated with the nuclear envelope and are involved in the regulation of nuclear structure and functions such as chromatin organization and gene transcription. Finally, we will test these methods on select targets from additional trypanosome organelles, to confirm they are broadly applicable, and ensure transferability to collaborating laboratories. With the wealth of information available from genome sequences and protein databases, we believe the methods we seek to develop will be a useful addition not only to proteomic studies in trypanosomes, but ultimately to other parasitic protists. PUBLIC HEALTH RELEVANCE: Trypanosomes cause devastating diseases in humans such as sleeping sickness. We are working to develop more efficient tools with which to study the protein interactions that are fundamental to the survival of these parasites. We believe that our methods will ultimately lead to the discovery of new drug targets to combat these disease-causing parasites.