Centrosomal Proteins in Giardia Differentiation

描述（由申请人提供）：蓝氏贾第鞭毛虫对外界刺激反应的分化能力是其发病机制的核心。贾第鞭毛虫细胞骨架在成囊和脱囊过程中发生了剧烈的变化。然而，介导这些关键转化的途径知之甚少。统一这一修订后的更新的总体假设是，中心体是细胞的“控制中心”，而贾第虫特有的细胞骨架元素具有特定的效应器功能，反映在它们的蛋白质组成。我们将用这些特定的目的来检验我们的假设：在包囊形成过程中，运动器官被内化。在具体目标1A，我们测试的假设，包囊需要定量和定性的变化，在蛋白质组成的贾第虫中心体/基体，指向新的水平的监管。本研究将利用多维蛋白质鉴定技术（MudPIT）对生长期和成囊期滋养体的中心体蛋白质组进行比较。在特定目标1B中，我们将在贾第虫中表位标记和表达选定的蛋白质，以验证它们的定位并用于目标2和3中的功能分析。细胞生物学研究的一个主要目标是将蛋白质定位的变化与分化的生理刺激联系起来。目前发现的大多数中心体蛋白也定位于贾第虫特有的细胞骨架元件。具体目标2是检验某些蛋白质定位于中心体或贾第虫特异性结构对生理信号有响应的假设。具体目标3是分析选定的中心体信号蛋白和贾第虫特有蛋白的生物学作用。在目标3A中，我们将鉴定与信号蛋白形成复合物的蛋白质，以检验它们通过与特定靶向或调节亚基结合而定位和发挥功能的假设。在目标3B中，我们将使用转录后基因沉默（PTGS）和过表达，通过定义所选信号传导和独特蛋白质在生长和分化中的功能来进一步检验这一假设。与公共卫生的相关性：蓝氏贾第鞭毛虫是美国和世界范围内水传播性腹泻病的主要原因，也是B级生物恐怖主义制剂。这些研究将极大地促进我们对G. Lamblia，其发病机制取决于其通过包囊或脱囊对来自其环境的变化信号快速响应的能力。贾第鞭毛虫是其他生命周期尚未完成的寄生虫的有价值的模型。

DESCRIPTION (provided by applicant): The ability of Giardia lamblia to differentiate in response to external stimuli is central to its pathogenesis. The Giardia cytoskeleton undergoes drastic changes during encystation and excystation. However, the pathways mediating these key transformations are poorly understood. The overall hypothesis unifying this revised renewal is that the centrosomes are cellular "control centers", while cytoskeletal elements unique to Giardia have specific effector functions that are reflected in their protein compositions. We will test our hypothesis with these Specific Aims: During encystation, the motility apparatus is internalized. In Specific Aim 1 A, we test the hypothesis that encystation entails quantitative and qualitative changes in the protein composition of Giardia centrosomes/basal bodies that point to new levels of regulation. We will compare the proteomes of centrosomes from growing and encysting trophozoites by Multidimensional Protein Identification Technology (MudPIT). In Specific Aim 1B, we will epitope-tag and express selected proteins in Giardia to validate their localization and for functional analyses in Aims 2 and 3. A major goal of cell biology research is to relate changes in protein localization to physiologic stimuli of differentiation. Most centrosomal proteins identified so far also localize to cytoskeletal elements that are unique to Giardia. Specific Aim 2 is to test the hypothesis that the localization of certain proteins to centrosomes or Giardia-specific structures is responsive to physiologic signals. Specific Aim 3 is to analyze the biological roles of selected centrosomal signaling proteins and proteins unique to Giardia. In Aim 3A, we will identify proteins that form complexes with signaling proteins to test the hypothesis that they localize and function by association with specific targeting or regulatory subunits. In Aim 3B, we will use post-transcriptional gene silencing (PTGS) and over- expression to further test this hypothesis by defining the functions of selected signaling and unique proteins in growth and differentiation. Relevance to public health: Giardia lamblia is a major cause of water-borne diarrheal disease in the US and worldwide and a Class B bioterrorism agent. These studies will greatly advance our understanding of the biology of G. lamblia, whose pathogenesis depends on its ability to respond rapidly to changing signals from its environment by encysting or excysting. Giardia is a valuable model for other parasites whose life cycles have not been completed.