Mechanisms of Giardia Iamblia Excystation

描述（由申请人提供）：兰氏贾第鞭毛虫是一种重要的人类病原体，必须成功脱壳才能感染新的宿主。由于细胞的清除速度太快，无法完全依赖于新的基因表达，因此该提议的统一假设是，第二信使途径可能是调控的核心。虽然我们可以在体外复制整个生命周期，但无论是精确的刺激还是调节细胞凋亡的第二信使途径都是未知的。基于广泛的初步数据，我们提出了以下具体目标：具体目标1是确定关键的生理分泌刺激，并验证鞭虫囊对这些信号的反应是由胞质钙和/或环AMP的变化介导或反映的假设。我们将研究“笼化”ca2v和/或cAMP进入胞质是否可以绕过相应的分泌刺激。具体目标2是定义钙调素（CAM）在调节细胞凋亡中的作用，并确定执行其功能的特定“下游”效应物。gCaM与蛋白激酶A共同定位于鞭毛基小体-中心体，提示在协调这些途径中起关键作用。我们将使用体内交联和基因组数据来功能鉴定在切除过程中与CaM特异性相互作用的下游效应物。特异性目的3是验证与从头蛋白质合成相比，蛋白质磷酸化的变化可能在调节细胞增生中起重要作用的假设。我们将比较蛋白质磷酸化（“磷酸化蛋白质组”）的变化与排出细胞中蛋白质表达的总体变化。序列分析和基因组数据将使我们能够确定关键的脱落蛋白。这种蛋白质组学方法将检验我们的整体假设，并补充Aims 1和Aims 2的细胞和分子数据。我们提出的研究将揭示重要的见解调控G - lambla增生。许多其他寄生虫具有囊性形式，其排泄是传播所必需的。贾第鞭毛虫可能是了解肠道寄生虫分化的一个有价值的模型。在基本水平上，贾第鞭毛虫增生是一种独特的模式，细胞从休眠中唤醒响应环境信号在早期分化的真核生物。

DESCRIPTION (provided by the applicant): Giardia lamblia, an important human pathogen, must successfully excyst in order to infect a new host. Since excystation is too rapid to rely entirely on new gene expression, the unifying hypothesis of this proposal is that second messenger pathways may be central for regulation. While we can reproduce the entire life cycle in vitro, neither the precise stimuli nor the second messenger pathways that regulate excystation are known. Based on extensive Preliminary Data, we propose these Specific Aims: Specific Aim 1 is to define the key physiological excystation stimuli, and to test the hypothesis that Giardia cysts' responses to these signals are mediated by or reflected in changes in cytosolic calcium and/or cyclic AMP. We will ask whether pharmacological release of "caged" Ca 2v and/or cAMP into the cytosol can bypass the corresponding excystation stimulus. Specific Aim 2 is to define the role(s) of calmodulin (CAM) in regulating excystation and to identify specific "downstream" effectors that carry out its function. gCaM co-localizes with protein kinase A to the flagellar basal bodies-centrosomes, suggesting a key role in coordinating these pathways. We will use in vivo cross-linking and genomic data to functionally identify downstream effectors that specifically interact with CaM during excystation. Specific Aim 3 is to test the hypothesis that changes in protein phosphorylation may be important in regulating excystation, compared with de novo protein synthesis. We will compare changes in protein phosphorylation (the "phosphoproteome"), with overall changes in the protein expression in excysting cells. Sequence analyses and genomic data will permit us to identify key excystation proteins. This proteomic approach will test our overall hypothesis and complement the cellular and molecular data of Aims 1 and 2. Our proposed studies will reveal important insights into regulation of G lamblia excystation. Many other parasites have cystic forms whose excystation is required for transmission. Giardia may be a valuable model for understanding parasite differentiation in the intestinal tract. On a basic level, Giardia excystation is a unique model for cellular awakening from dormancy in response to environmental signals in an early-diverging eukaryotes.