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）：贾第鞭毛虫病是由真核寄生虫兰氏贾第鞭毛虫引起的疾病，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 亿贾第鞭毛虫病例。新出现的耐药菌株被证明很难治疗，而一线治疗药物甲硝唑的副作用发生率很高，因此迫切需要。针对新分子途径的抗贾第鞭毛虫药物属于最早分化的真核生物群体之一，因此与后生动物有很大不同，该提案探讨了 Rho 家族 GTP 酶的生物学特性。这些蛋白质作为控制重要细胞过程的分子开关，含有一个 Rho 家族 GTP 酶同源物 gRac，此前已被证明发挥着保守的作用。 gRac 在调节极性、膜运输和细胞骨架中发挥着重要作用，所有这些对于活力和发病机制都至关重要。 ) ) 产生耐环境包囊，是贾第鞭毛虫中唯一已知的调节分泌途径。贾第鞭毛虫中的触发仍然是个谜，但 gRac 可能是调节这一过程的候选者，该提案的目标 1 通过跟踪囊壁标记通过分泌过程来确定 gRac n 囊肿感染形成的具体作用。对于下游过程，使用亲和纯化和质谱捕获先前未识别的下游效应器（贾第鞭毛虫特异性或可能古老且未被发现），将 gRac 信号传导与细胞骨架和膜运输联系起来。目标 2 确定了 199 个假定的 gRac 相互作用因子，建议通过显微镜和敲低研究验证其中的 4 个 gRac 相互作用因子作为膜运输和/或细胞骨架的下游效应子，gRac 的上游是一组用于激活或抑制的 gRac 调节剂。 gRac 信号传导。目标 3，建议结合消耗研究和确定它们对 gRac 信号传导的贡献，并确定每种调节剂的特定生物学作用。测量细胞增殖、寄生虫附着和形成感染性包囊的能力的实验预计将确定 gRac 在贾第鞭毛虫生物学中的核心作用，并为发现新的治疗靶点铺平道路。该系统仅包含一个 Rho GTP 酶和少量上游调节剂，因此非常容易处理，并且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础生物学。