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-原生动物寄生虫的研究，锥虫在发展中国家引起重大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的所有蛋白质进行表征。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几乎不可或缺的工具。然而，这两种方法都需要适当的样品制备来产生高质量的结果，并且真核基因组编码的蛋白质的绝对数量意味着需要高通量方法来快速和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研究酵母的锥虫研究的技术，例如：（i）冷冻溶解，一种用于裂解冷冻细胞以保存蛋白复合物的方法，因为它们在收集时;（ii）96孔高通量筛选，以确定任何蛋白复合物的最佳缓冲条件，在快速和简便的程序中使用最少量的细胞材料;（iii）随机或靶向相互作用的同位素区分（I-DIRT）-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经证实的方法。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）的选定数量的组分蛋白（称为NUPS）作为测试床。NPC是细胞核和细胞质之间交换的唯一介质;每个NPC是由30个不同的NUP组成的约50 MDa的大分子组装体，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（因为它参与核转运，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），也因为我们以前在布氏锥虫（TbNups）中鉴定和GFP标记的22个Nups，因此现在所有Nups都携带方便的亲和手柄。我们将首先使用所有已识别的TbNups优化方法。然后，我们将专注于TbNup 92，Nup在有丝分裂期间重新定位到纺锤体组织者。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，细胞周期调节的过程。接下来，我们将扩展这些蛋白质组学工具来探索纤层;核内的细丝网络，与核膜密切相关，并参与核结构和功能的调节，如染色质组织和基因转录。最后，我们将在其他锥虫细胞器的选定目标上测试这些方法，以确认它们具有广泛的适用性，并确保可转移到合作实验室。随着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是一个有用的除了蛋白质组学研究锥虫，但最终其他寄生原生生物。 公共卫生相关性：锥虫会引起人类的毁灭性疾病，如昏睡病。我们正在努力开发更有效的工具来研究这些寄生虫生存所必需的蛋白质相互作用。我们相信，我们的方法将最终导致发现新的药物靶点，以打击这些致病寄生虫。

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.