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（由适用提供）：贾diasis是由真核生物寄生虫贾迪亚兰布利亚（Giardia Lamblia）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；估计表明每年有2.8亿例贾第鞭毛虫病例。新出现的耐药品菌株正在提供难以治疗的菌株，甲硝唑的一线治疗具有很高的副作用。因此，对靶向新分子途径的抗基亚迪亚药物的迫切需要。贾第鞭毛虫属于最早的真核生物群体之一，因此与后生动物相差很大。在这种不同的生物学中，存在开发靶向治疗的机会。该提案探讨了贾第藻中Rho家族GTPases的生物学。这些蛋白充当控制必需细胞过程的分子开关。 Giardia包含一个单一的Rho家族GTPase同源物GRAC，以前证明在调节的极性，膜运输和细胞骨架中起着配置的作用，所有这些都对生存能力和发病机理必不可少。功能测定表明，GRAC在囊肿形成中起作用。此过程需要囊肿壁蛋白（CWP）的脉冲生产，加工和分泌才能使环境抗性囊肿，并且是贾第藻中唯一已知的调节分泌途径。已知Rho GTPases会调节植物和动物中的Er-Golgi运输和秘密事件。在贾第鞭毛虫中触发CWP的分泌如何仍然是神秘的，但GRAC可能是确定这一过程的候选人。该提案的目标1通过通过秘密过程遵循囊肿壁标记来确定Grac N感染性囊肿形成的特定作用。还未知的是GRAC信号如何与下游过程相关联，以前未鉴定的下游效应（贾第鞭毛虫特异性或潜在的古老和未发现）是将GRAC信号与细胞骨架和膜贩运联系起来的。使用亲和力纯化和质谱法，确定了199个推定的GRAC相互作用者。目标2通过显微镜和敲低研究来验证这些GRAC相互作用的四个相互作用者作为膜运输和/或细胞骨架的下游效应。 GRAC的上游是一组六个GRAC调节剂，可激活或抑制GRAC信号传导。 AIM 3，提案，以确定其对GRAC信号传导的贡献，并使用部署研究和测定的组合来确定每个调节剂的特定生物学作用，以测量细胞增殖，寄生虫的附着以及形成感染性囊肿的能力。提出的实验预计将定义GRAC在贾第鞭毛虫生物学中的核心作用，并为发现新型治疗靶标的发现铺平了道路。此外，由于贾第鞭毛虫的基因组仅包含一个Rho GTPase和少数上游调节剂，因此该系统是高度易处理的，可能会揭示基本生物学，而基本生物学已被模型真核生物中发现的更复杂的信号系统所掩盖。