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（由申请人提供）：血管炎症在动脉粥样硬化等疾病中起着关键作用。血管炎症由几个离散的阶段组成，第一阶段由内皮细胞胞吐作用驱动。该应用的目标是识别调节内皮胞吐作用的新型细胞因子。 囊泡运输是对细胞生物学至关重要的过程。在胞吐作用期间，囊泡将蛋白质从高尔基体递送到细胞外部。几个蛋白质家族已被确定为介导胞吐作用，但我们对胞吐机制的了解是零散且不完整的。我们不知道：（1）细胞如何调节运输囊泡的数量，（2）出芽的囊泡如何与供体室分离，或（3）囊泡如何靶向其正确的目的地。胞吐途径中新蛋白质的鉴定可能有助于回答其中一些基本问题。 该领域取得进展的一个关键障碍在于历史实验模型。我们关于运输的大部分知识都依赖于对分泌神经囊泡的神经元的研究。然而，神经囊泡的胞吐作用的研究具有挑战性：神经递质的测定既不快速也不稳健。相比之下，内皮细胞将血管性血友病因子分泌到培养基中，这是一种易于以高通量形式测量的蛋白质。 我们假设一组离散的蛋白质调节内皮颗粒的胞吐作用。为了检验这一假设，我们计划鉴定和表征参与胞吐作用的蛋白质，具体目标如下：目标#1：鉴定通过调节内皮胞吐作用的新蛋白质 功能基因组筛选。我们将进行大规模 RNA 干扰筛选，以确定调节内皮细胞胞吐作用的蛋白质和途径。在排除影响细胞活力的基因后，我们将验证增加或减少内皮胞吐作用的单个基因。 目标#2：表征调节内皮细胞胞吐作用的新型蛋白质的作用。接下来我们将描述一些已确定的候选基因产物的特征 目标#1。我们将确定这些候选蛋白影响囊泡运输的哪个阶段。我们将研究候选基因产物在颗粒生物合成、货物装载、囊泡运输和膜融合中的作用。此外，我们还将研究我们的顾问之前在全基因组关联研究中发现的 5 个候选基因，这些基因与人类内皮胞吐作用的改变有关。 该提议的意义在于，囊泡运输途径很重要但尚未被充分理解，并且它们对于动脉粥样硬化等人类血管炎症性疾病至关重要。 该提案的创新点在于（1）鉴定了调节囊泡运输的新基因，（2）新基因的表征 最近在人类中发现了与胞吐作用相关的产物，（3）识别人类细胞中胞吐蛋白的新方法。