Rac: a critical regulator of the cytoskeleton and membrane trafficking in Giardia

Rac：贾第鞭毛虫细胞骨架和膜运输的关键调节因子

• 批准号: 8884918
• 负责人: Alexander Richard Paredez
• 金额: $ 38.63万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-02-01 至 2020-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8884918
• 关键词: Actin-Binding Protein; Actins; Adverse effects; Affect; Affinity Chromatography; Animals; Architecture; Biogenesis; Biological; Biological Assay; Biology; CDC42 gene; Cell Line; Cell Proliferation; Cell Survival; Cell Wall; Cell physiology; Cells; Child; Complex; Coupled; Cyst; Cytokinesis; Cytoskeletal Proteins; Cytoskeleton; Defect; Deposition; Diarrhea; Disease; Drug Targeting; Drug resistance; Electronics; Endocytosis; Ensure; Eukaryota; Event; Exocytosis; Family; Genome; Giardia; Giardia lamblia; Giardiasis; Golgi Apparatus; Guanosine Triphosphate Phosphohydrolases; Homologous Gene; Human; Incidence; Intestinal parasite; Life Cycle Stages; Link; Liquid Chromatography; Malabsorption Syndromes; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Measures; Membrane; Membrane Protein Traffic; Metronidazole; Microscopy; Microtubules; Modeling; Molecular; Monitor; Morbidity - disease rate; Organelles; Parasites; Parasitic Intestinal Diseases; Pathogenesis; Pathogenicity; Pathway interactions; Physiologic pulse; Plants; Play; Process; Production; Protein Secretion; Proteins; Regulation; Relative (related person); Research Personnel; Resistance; Role; Signal Transduction; Staging; Stream; System; Testing; Translations; Transmembrane Transport; Vesicle; base; cell motility; gastrointestinal; inhibitor/antagonist; new therapeutic target; novel; protein transport; public health relevance; research study; resistant strain; rho; rho GTP-Binding Proteins; screening; secretion process; secretory protein; spastin; tandem mass spectrometry; therapeutic target; transmission process

 DESCRIPTION (provided by applicant): Giardiasis, the disease caused by the eukaryotic parasite Giardia lamblia, is the most common intestinal parasitic disease in the U.S. and a major cause of morbidity in children throughout the world; estimates indicate 280 million cases of Giardia annually. Newly emerging drug-resistant strains are proving difficult to treat, and the front-line treatment, metronidazole, has a high incidence of side effects. Therefore, there is a critical need for anti-Giardia drugs that target novel molecular pathways. Giardia belongs to one of the earliest diverging groups of eukaryotes and is therefore quite divergent from metazoans. Opportunities for developing targeted therapeutics exist within this divergent biology. This proposal explores the biology of Rho family GTPases in Giardia. These proteins act as molecular switches that control essential cellular processes. Giardia contains a single Rho family GTPase homolog, gRac, previously demonstrated to play a conserved role in regulating polarity, membrane trafficking, and the cytoskeleton, all of which are essential to viability and pathogenesis. Functional assays have demonstrated that gRac plays a role in cyst formation. This process requires pulsed production, processing, and secretion of cyst wall protein (CWP) to make environmentally resistant cysts and is the only known regulated secretory pathway in Giardia. Rho GTPases are known to regulate ER-Golgi transport and secretory events in plants and animals. How CWP secretion is triggered in Giardia remains enigmatic, yet gRac is a likely candidate for regulating this process. Aim 1 of this proposal identifies the specific role of gRac n infectious cyst formation by following cyst wall markers through the secretory process. Also unknown is how gRac signaling is linked to downstream processes, previously unidentified downstream effectors (Giardia-specific or potentially ancient and undiscovered) are hypothesized to link gRac signaling to the cytoskeleton and membrane trafficking. Using affinity purification and mass spectroscopy, 199 putative gRac interactors were identified. Aim 2 proposes to validate four of these gRac interactors as downstream effectors of membrane trafficking and/or the cytoskeleton through microscopy and knockdown studies. Upstream of gRac is a set of six gRac modulators that act to activate or inhibit gRac signaling. Aim 3, proposes to determine their contribution to gRac signaling and identify specific biological roles for each modulator using a combination of depletion studies and assays to measure cell proliferation, parasite attachment, and the ability to form infectious cysts. The proposed experiments are expected to define the central role of gRac in Giardia biology and pave the way toward discovery of novel therapeutic targets. Furthermore, because the Giardia genome contains only a single Rho GTPase and a handful of upstream modulators, the system is highly tractable and may uncover fundamental biology that has been obscured by the more complex signaling systems found in model eukaryotes.

 描述（由申请人提供）：贾第虫病是由真核寄生虫贾第虫引起的疾病，是美国最常见的肠道寄生虫病，也是全世界儿童发病的主要原因;估计每年有2.8亿例贾第虫病。新出现的耐药菌株被证明难以治疗，而一线治疗甲硝唑的副作用发生率很高。因此，迫切需要靶向新分子途径的抗贾第虫药物。贾第鞭毛虫属于真核生物最早分化的类群之一，因此与后生动物非常不同。在这种不同的生物学中存在开发靶向治疗的机会。该提案探讨了贾第虫Rho家族GTP酶的生物学。这些蛋白质充当控制基本细胞过程的分子开关。贾第虫含有一个单一的Rho家族GTdR同源物，gRac，以前被证明在调节极性，膜运输和细胞骨架中发挥保守作用，所有这些都是生存力和发病机理所必需的。功能测定已经证明gRac在囊肿形成中起作用。这一过程需要脉冲产生、加工和分泌囊壁蛋白（CWP），以产生耐环境的囊，是贾第虫中唯一已知的受调节的分泌途径。已知Rho GTP酶调节植物和动物中的ER-高尔基体运输和分泌事件。在贾第虫中如何触发CWP分泌仍然是个谜，但gRac是调节这一过程的可能候选者。该提议的目的1通过在整个分泌过程中跟踪囊壁标志物来鉴定gRac在感染性囊肿形成中的特定作用。同样未知的是gRac信号传导如何与下游过程相关联，假设先前未鉴定的下游效应物（贾第虫特异性或潜在的古老和未发现的）将gRac信号传导与细胞骨架和膜运输相关联。使用亲和纯化和质谱，鉴定了199种推定的gRac相互作用物。目的2提出通过显微镜和敲除研究来验证这些gRac相互作用物中的四种作为膜运输和/或细胞骨架的下游效应物。gRac的上游是一组六种gRac调节剂，其用于激活或抑制gRac信号传导。目的3，提出确定它们对gRac信号传导的贡献，并使用消耗研究和测定的组合来确定每种调节剂的特定生物学作用，以测量细胞增殖、寄生虫附着和形成感染性包囊的能力。预计拟议的实验将确定gRac在贾第虫生物学中的核心作用，并为发现新的治疗靶点铺平道路。此外，由于贾第虫基因组仅包含一个Rho GTdR和少数上游调节剂，因此该系统非常易于处理，并可能揭示被模型真核生物中发现的更复杂的信号系统所掩盖的基本生物学。