Phosphoinositides in the T. brucei Endomembrane System

布氏锥虫内膜系统中的磷酸肌醇

• 批准号: 8687963
• 负责人: Julia K Gilden
• 金额: $ 5.51万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-15 至 2016-09-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8687963
• 关键词: Address; Adverse effects; Affinity; Affinity Chromatography; Africa South of the Sahara; African Trypanosomiasis; Antibodies; Area; Autophagocytosis; Back; Binding; Binding Proteins; Biological Assay; Biology; Biosensor; Blast Cell; Blood Circulation; Cattle; Cell Extracts; Cell Nucleus; Cell membrane; Cells; Cellular biology; Cessation of life; Country; Development; Disease; Early Endosome; Electron Microscopy; Endocytosis; Endosomes; Eukaryota; Exocytosis; Foundations; Future; Genome; Goals; Golgi Apparatus; Human; Hydrolase; Image; Immunofluorescence Immunologic; Individual; Inositol; Investigation; Lipids; Liposomes; Liquid substance; Livestock; Lysosomes; Lytic; Maps; Mass Spectrum Analysis; Measures; Membrane; Membrane Glycoproteins; Membrane Proteins; Methods; Nutrient; Organelles; Organism; Parasites; Pathway interactions; Peptide Hydrolases; Phagocytosis; Pharmaceutical Preparations; Phase; Phosphatidylinositols; Phosphoric Monoester Hydrolases; Phosphotransferases; Positioning Attribute; Process; Production; Proteins; Protozoa; Public Health; RNA Interference; Recycling; Resolution; Role; Signal Transduction; Sorting - Cell Movement; Surface; System; Therapeutic; Transferrin Receptor; Treatment Protocols; Trypanosoma; Trypanosoma brucei brucei; Tsetse Flies; Two-Dimensional Gel Electrophoresis; Vaccination; Variant; Vesicle; Work; Yeasts; cell fixing; drug development; feeding; in vitro Assay; late endosome; light microscopy; nagana; novel; programs; public health relevance; rab GTP-Binding Proteins; receptor binding; trafficking; uptake; vector control

DESCRIPTION (provided by applicant): Trypanosoma brucei ssp. are parasitic protozoa responsible for causing human African trypanosomiasis (HAT, sleeping sickness) in humans and nagana in cattle and other livestock. The parasite is endemic to 36 countries in sub-Saharan Africa, where tsetse fly vector control programs have been the major factors in the recent decline of the disease. Still, the disease is invariably fatal when untreated, and the few drugs available to treat HAT have serious limitations such as prolonged treatment regimen, expense, difficult storage requirements, and serious side effects including death. Understanding the basic cell biology of T. brucei is essential to the development of better drugs that will contribute to the WHO goal of total elimination of HAT as a public health problem. One aspect of T. brucei biology that could be exploited for the development of drugs is the trafficking of endocytic cargo. Bloodstream form trypanosomes have an unusually streamlined endomembrane system that allows for very rapid uptake and recycling or degradation of material. Some aspects of this system have been explored, such as the necessity for small Rab GTPases in regulating different compartments. In other eukaryotes, multiple steps in the endocytic pathway are regulated by signaling lipids called phosphoinositides. Phosphatidylinositol can be phosphorylated on positions 3,4, and 5 of the inositol ring to form seven distinct species. Through the localized action of kinases and phosphatases, individual phosphoinositides are restricted to specific membranes. Enrichment of specific phosphoinositides can therefore mark the identity of functional regions or membrane-bound compartments. Downstream functions arise from the subsequent recruitment of effector proteins, which bind to phosphoinositides through conserved domains including PH, PX, and FYVE domains. PI(3)P and PI(3,5)P2 have particularly been implicated in trafficking between endocytic organelles. Despite their importance in other systems, very little is known about phosphoinositides in T. brucei. This proposal seeks to specifically explore the roles of PI(3)P and PI(3,5)P2 in T. brucei endocytic trafficking. We intend to use biosensors to map the subcellular localization of those phosphoinositides by light and electron microscopy. Next, we will use a knockdown approach to disrupt production of PI(3)P and PI(3,5)P2 independently and define their roles in endocytic trafficking. Finally, we will use an affinity purification method t identify potential effector proteins of PI(3)P and PI(3,5)P2. This work will raise many new questions in T. brucei cell biology and potentially lay the groundwork for future drug development exploiting this pathway.

描述（由申请人提供）：Brucei SSP锥虫。是寄生的原生动物负责在人类和其他牲畜中引起人类和长纳纳的人类非洲锥虫病（帽子，睡眠疾病）。该寄生虫在撒哈拉以南非洲的36个国家中是特有的，在该疾病近期下降，采摘蝇载体控制计划一直是该疾病下降的主要因素。尽管如此，在未经治疗的情况下，这种疾病总是致命的，并且可以治疗帽子的少数药物具有严重的局限性，例如长时间的治疗方案，费用，困难的存储要求以及包括死亡在内的严重副作用。了解布鲁氏菌的基本细胞生物学对于开发更好的药物至关重要，这将有助于将HAT完全消除作为公共卫生问题的WHO目标。可以利用用于开发毒品的布鲁氏菌生物学的一个方面是贩运内吞货物。血液形式的锥虫具有异常流线型的内膜系统，可快速摄取和回收或降解材料。已经探索了该系统的某些方面，例如在调节不同隔室时需要小型RAB GTPase的必要性。在其他真核生物中，内吞途径中的多个步骤受到称为磷酸肌醇的信号脂质的调节。磷脂酰肌醇可以在3,4的位置和5个肌醇环上磷酸化，以形成七个不同的物种。通过激酶和磷酸酶的局部作用，单个磷酸肌醇仅限于特定的膜。因此，特定磷酸肌醇的富集可以标记功能区域或膜结合室的身份。下游功能是由随后的效应蛋白的募集引起的，效应蛋白通过pH，PX和FYVE结构域（包括pH，PX和FYVE结构域）与磷酸肌醇结合。 PI（3）P和PI（3,5）P2特别与内吞细胞器之间的运输有关。尽管它们在其他系统中的重要性，但对布鲁氏菌中的磷酸肌醇知之甚少。该提案旨在专门探索PI（3）P和PI（3,5）P2在Brucei内吞贩运中的作用。我们打算使用生物传感器通过光和电子显微镜来绘制这些磷酸肌醇的亚细胞定位。接下来，我们将使用敲低方法独立地破坏PI（3）P和PI（3,5）P2的产生，并定义其在内吞贩运中的作用。最后，我们将使用亲和力纯化方法t识别PI（3）P和PI（3,5）P2的潜在效应蛋白。这项工作将在T. Brucei细胞生物学中提出许多新问题，并有可能为利用这一途径的未来药物开发奠定基础。