Role of Phospholipid Remodeling in Secretion and Golgi Function in Mammalian Cell

磷脂重塑在哺乳动物细胞分泌和高尔基体功能中的作用

• 批准号: 8437327
• 负责人: WILLIAM J BROWN
• 金额: $ 29.76万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-19 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8437327
• 关键词: Acyltransferase; Animals; Atherosclerosis; Basic Science; Binding; Biological; Biological Process; Caliber; Carrier Proteins; Cell membrane; Cells; Coated vesicle; Complex; Cystic Fibrosis; Defect; Digestion; Disease; Enzymes; Event; Genetic; Goals; Golgi Apparatus; Grant; Health; Hormones; Human; Intracellular Membranes; Laboratories; Length; Lysophospholipids; Malignant Neoplasms; Mammalian Cell; Mediating; Membrane; Membrane Protein Traffic; Molecular; Movement; Mutation; Nerve Degeneration; Organelles; Organism; Pathway interactions; Phospholipase A2; Phospholipases A; Phospholipids; Physiological Processes; Play; Process; Production; Role; Shapes; Signal Transduction; Small Interfering RNA; Structure; Vesicle; Work; human disease; in vitro Assay; in vivo; neurotransmission; novel; pathogen; public health relevance; reconstitution; research study; secretion process; trafficking

DESCRIPTION (provided by applicant): This grant focuses on the basic cell biological process of membrane trafficking through the secretory pathway in mammalian cells. Secretion plays fundamental biological roles at the level of both single cells and multi-cellular organisms t control a wide array of physiological processes including release of hormones and digestive enzymes, neurotransmission, and defense against foreign pathogens. Secretion involves the transport of cargo between intracellular organelles, e.g., between the Golgi complex and plasma membrane. Defects in the transport of proteins through these pathways are associated with a host of diseases including cancer, cystic fibrosis, neurodegeneration, and atherosclerosis. In mammalian cells, membrane tubules (60-80 nm in diameter and up to many microns in length) emanate from various organelles of the secretory pathway, and these tubules have been implicated in many intracellular trafficking steps, e.g., retrograde trafficking from the Golgi complex to the ER and anterograde movement through the Golgi stack. Recent evidence from my laboratory indicates that cytoplasmic phospholipase A (PLA) and integral membrane lysophospholipid acyltransferase (LPAT) enzymes, with opposing enzymatic activities, work in concert to mediate membrane tubule formation by directly altering membrane phospholipid composition and consequently membrane shape. The long-term objectives of this grant are to elucidate the roles of membrane tubules in intracellular trafficking, and to determine the biological functions of specific PLA and LPAT enzymes with respect to secretion in mammalian cells. The goals of this work are encompassed in three Specific Aims, with a focus on the mammalian Golgi complex: 1) identify and characterize PLA and LPAT enzymes involved in intracellular membrane trafficking; 2) determine the contribution of PLA2 and LPAT enzymes to the production of Golgi coated vesicles and membrane tubules; and 3) determine the molecular mechanisms by which PLA2 and LPATs contribute to membrane tubule formation. These goals will be achieved through a combination of molecular, cellular, and genetic approaches. The in vivo roles of candidate PLA and LPAT enzymes in secretory trafficking will be investigated by conducting over expression and siRNA knockdown experiments. These experiments will focus on membrane trafficking to, through, and from the Golgi complex, and vesicle fission from membrane-bound organelles. In vitro assays that reconstitute tubule or vesicle formation will also be conducted to elucidate the mechanisms underlying the effects of PLA and LPAT activity on membrane shape. These studies will reveal novel biological roles for PLA and LPAT enzymes in mediating intracellular trafficking events in the secretory pathway and advance our understanding of membrane tubule-mediated transport.

描述（由申请人提供）：该赠款重点是通过哺乳动物细胞中分泌途径的膜运输的基本细胞生物学过程。分泌在单细胞和多细胞生物的水平上扮演基本的生物学作用，t控制着各种各样的生理过程，包括释放激素和消化酶，神经传递，以及针对异物病原体的防御。分泌涉及在细胞内细胞器之间的运输，例如高尔基体络合物和质膜之间。蛋白质通过这些途径运输的缺陷与许多疾病有关，包括癌症，囊性纤维化，神经变性和动脉粥样硬化。在哺乳动物细胞中，膜小管（直径60-80 nm，长度长达多个微米长）来自分泌途径的各种细胞器，这些小管与许多细胞内贩运步骤有关，例如，从高尔基复合体到eR和golgi的运动中逆行，从逆转行动中进行了逆行。我的实验室的最新证据表明，具有相反的酶促活性的胞质磷脂酶A（PLA）和整体膜溶质磷脂酰基转移酶（LPAT）酶，可以通过直接改变膜磷脂组成和相关的膜状膜状膜状形状来介导膜浴缸形成。该赠款的长期目标是阐明膜小管在细胞内运输中的作用，并确定特定PLA和LPAT酶在哺乳动物细胞中分泌方面的生物学功能。这项工作的目标包含在三个特定目标中，重点是哺乳动物高尔基体综合体：1）识别和表征与细胞内膜运输有关的PLA和LPAT酶； 2）确定PLA2和LPAT酶对高尔基涂层囊泡和膜小管的产生的贡献； 3）确定PLA2和LPAT有助于膜小管形成的分子机制。这些目标将通过分子，细胞和遗传方法的结合来实现。 候选PLA和LPAT酶在分泌贩运中的体内作用将通过进行表达和siRNA敲低实验来研究。这些实验将集中于膜运输，通过高尔基体复合物以及膜结合细胞器的囊泡裂变。还将进行重新构造小管或囊泡形成的体外测定，以阐明PLA和LPAT活性对膜形状的影响的基础机制。这些研究将揭示PLA和LPAT酶在介导分泌途径中的细胞内贩运事件中的新型生物学作用，并促进我们对膜小管介导的转运的理解。