The development of tools for proteomic analysis in trypanosomes

描述(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。锥虫是在发展中国家造成重大公共卫生和经济问题的原生动物寄生虫。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中的所有蛋白质进行表征。亲和纯化和质谱学是常规用于鉴定蛋白质的强大工具，已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准备才能产生高质量的结果，而真核基因组编码的蛋白质的绝对数量意味着需要高通量的方法来快速和系统地分析单个蛋白质所产生的所有可能的相互作用。出于这些原因，我们正在调整和开发用于酵母研究的技术，以用于锥体研究，例如：(I)冷冻裂解，一种裂解冷冻细胞以保存其收集时的蛋白质复合体的方法；(Ii)96孔高通量筛选，以使用最少的细胞材料快速和简便地确定任何蛋白质复合体的最佳缓冲条件；(Iii)随机或靶向相互作用的同位素差异(I-污垢)-这是一种成熟的使用稳定同位素标记来区分特异和非特异性相互作用蛋白质的方法。为了在锥体中开发和验证这些方法，我们选择使用选定数量的核孔复合体(NPC)的组成蛋白(称为NUP)作为试验台。鼻咽癌是核质交换的唯一媒介；每个鼻咽癌是一个~50MDa的大分子集合体，由30个不同的核蛋白组成，总共有~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(因为它参与了核运输、核糖核蛋白复合体组装、细胞周期控制和染色质修饰复合体)，还因为我们以前在布氏锥虫(TbNup)中发现并标记了22个NUP，因此现在都带有一个方便的亲和柄。我们将首先使用所有确定的TbNup来优化方法。然后我们将关注TbNup92，一种在有丝分裂过程中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方法可以获得高度动态的、细胞周期调节的过程。接下来，我们将扩展这些蛋白质组学工具来探索板层；板层是细胞核内与核膜密切相关的细丝网络，参与核结构和功能的调节，如染色质组织和基因转录。最后，我们将在从更多锥虫细胞器中选择的靶标上测试这些方法，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中丰富的信息，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用，而且最终将对其他寄生性原生生物有用。

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.