Novel Regulators of Endothelial Exocytosis

内皮胞吐作用的新型调节剂

• 批准号: 8445932
• 负责人: CHARLES J LOWENSTEIN
• 金额: $ 26.86万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-01-15 至 2014-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8445932
• 关键词: Adherence; Affect; Americas; Area; Atherosclerosis; Biogenesis; Biological Assay; Biological Models; Blood Vessels; Candidate Disease Gene; Carrier Proteins; Cause of Death; Cell Survival; Cells; Cellular biology; Cytoplasmic Granules; Destinations; Development; Disease; Endothelial Cells; Exocytosis; Experimental Models; Future; Genes; Goals; Golgi Apparatus; Grant; Human; Individual; Inflammatory; Knowledge; Leukocyte Trafficking; Leukocytes; Measures; Mediating; Mediator of activation protein; Membrane Fusion; Modeling; Molecular Motors; Morbidity - disease rate; Neurons; Neurotransmitters; Pathway interactions; Play; Preparation; Process; Protein Family; Proteins; RNA Interference; Research; Research Personnel; Role; Scientist; Set protein; Shapes; Staging; Synaptic Vesicles; System; Testing; Therapeutic Agents; Thrombosis; Transgenic Mice; Transport Vesicles; United States; Vesicle; Yeasts; atherogenesis; design; editorial; functional genomics; genome wide association study; in vivo; injured; innovation; insight; mortality; motor control; novel; novel strategies; polypeptide; public health relevance; trafficking; vascular inflammation; von Willebrand Factor

DESCRIPTION (provided by applicant): Vascular inflammation plays a key role in diseases such as atherosclerosis. Vascular inflammation consists of several discrete stages, and the first stage is driven by endothelial cell exocytosis. The goal of this application is to identify novel cellular factors that regulate endothelial exocytosis. Vesicle trafficking is a process of fundamental importance to cell biology. During exocytosis, vesicles deliver proteins from the Golgi to the cell exterior. Several families of proteins have been identified as mediating exocytosis, but our knowledge of the exocytic machinery is fragmentary and incomplete. We do not know: (1) how cells regulate the number of transport vesicles, (2) how budding vesicles separate from the donor compartment, or (3) how vesicles are targeted to their correct destination. Identification of novel proteins in the exocytic pathway may help answer some of these fundamental questions. A critical barrier to progress in this field lies in historical experimental models. Most of our knowledge about transport relies on the study of neurons secreting neurovesicles. However, exocytosis of neurovesicles is challenging to study: assays for neurotransmitters are neither rapid nor robust. In contrast, endothelial cells secrete von Willebrand Factor into the media, a protein easy to measure in a high-throughput format. We hypothesize that a discrete set of proteins regulates exocytosis of endothelial granules. To test this hypothesis, we plan to identify and characterize proteins involved in exocytosis, in the following specific aims: Aim #1: Identify novel proteins that regulate endothelial exocytosis by a functional genomic screen. We will perform a large-scale RNA interference screen to identify proteins and pathways that modulate exocytosis in endothelial cells. After excluding genes that affect cell viability, we will validate individual genes that increase or decrease endothelial exocytosis. Aim #2: Characterize the role of novel proteins that regulate exocytosis in endothelial cells. Next we will characterize a select few of the candidate gene products identified in Aim #1. We will determine which stage of vesicle trafficking these candidate proteins affect. We will study the role of candidate gene products in granule biogenesis, cargo loading, vesicle trafficking, and membrane fusion. In addition, we will also study 5 candidate genes previously identified in a genome wide association study by our Consultant which are associated with altered endothelial exocytosis in humans. The significance of this proposal is that vesicle trafficking pathways are important but not well understood, and they are critical to human vascular inflammatory diseases such as atherosclerosis. The innovation of this proposal is (1) identification of novel genes that regulate vesicle trafficking, (2) characterization of novel gene products associated with exocytosis recently identified in humans, (3) new approaches to identifying exocytic proteins in human cells.

描述（申请人提供）：血管炎症在动脉粥样硬化等疾病中起着关键作用。血管炎症包括几个独立的阶段，第一阶段是由内皮细胞胞吐驱动的。这个应用程序的目的是确定新的细胞因子调节内皮胞吐。囊泡转运是细胞生物学的一个重要过程。在胞吐过程中，囊泡将蛋白质从高尔基体运送到细胞外部。已经确定了几个蛋白家族介导胞吐作用，但我们对胞吐机制的了解是零碎和不完整的。我们不知道：(1)细胞如何调节运输囊泡的数量，(2)出芽囊泡如何从供体隔室分离，或(3)囊泡如何靶向到正确的目的地。鉴别胞外通路中的新蛋白可能有助于回答这些基本问题。在这一领域取得进展的一个关键障碍在于历史实验模型。我们对运输的大部分知识依赖于对分泌神经囊泡的神经元的研究。然而，神经囊泡胞吐的研究具有挑战性：神经递质检测既不快速也不可靠。相反，内皮细胞分泌血管性血液病因子到培养基中，这是一种易于以高通量格式测量的蛋白质。我们假设一组离散的蛋白质调节内皮颗粒的胞吐。为了验证这一假设，我们计划在以下具体目标中鉴定和表征参与胞吐的蛋白质：目标#1：鉴定通过a