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）：布氏锥虫亚种。是寄生原生动物，可引起人类非洲锥虫病（HAT，昏睡病）以及牛和其他牲畜的那加那病。这种寄生虫是撒哈拉以南非洲 36 个国家的地方病，这些国家的采采蝇媒介控制计划是最近该疾病减少的主要因素。尽管如此，如果不治疗，这种疾病总是致命的，并且可用于治疗 HAT 的少数药物具有严重的局限性，例如治疗方案延长、费用昂贵、储存要求困难以及包括死亡在内的严重副作用。了解布氏锥虫的基本细胞生物学对于开发更好的药物至关重要，这将有助于实现世界卫生组织彻底消除 HAT 这一公共卫生问题的目标。布氏锥虫生物学可用于药物开发的一方面是内吞货物的贩运。血流形式的锥虫具有异常精简的内膜系统，可以非常快速地吸收和回收或降解材料。该系统的某些方面已被探索，例如小 Rab GTP 酶在调节不同区室中的必要性。在其他真核生物中，内吞途径中的多个步骤由称为磷酸肌醇的信号脂质调节。磷脂酰肌醇可在肌醇环的 3,4 和 5 位上磷酸化，形成七种不同的物质。通过激酶和磷酸酶的局部作用，单个磷酸肌醇被限制在特定的膜上。因此，特定磷酸肌醇的富集可以标记功能区域或膜结合区室的身份。下游功能由随后效应蛋白的募集产生，效应蛋白通过保守结构域（包括 PH、PX 和 FYVE 结构域）与磷酸肌醇结合。 PI(3)P 和 PI(3,5)P2 特别参与内吞细胞器之间的运输。尽管它们在其他系统中很重要，但人们对布氏锥虫中的磷酸肌醇知之甚少。该提案旨在专门探讨 PI(3)P 和 PI(3,5)P2 在 T. brucei 内吞运输中的作用。我们打算使用生物传感器通过光学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破坏 PI(3)P 和 PI(3,5)P2 的产生，并确定它们在内吞运输中的作用。最后，我们将使用亲和纯化方法来鉴定 PI(3)P 和 PI(3,5)P2 的潜在效应蛋白。这项工作将提出布氏锥虫细胞生物学中的许多新问题，并可能为未来利用该途径的药物开发奠定基础。