Novel Regulators of Endothelial Exocytosis

描述（由申请人提供）：血管炎症在动脉粥样硬化等疾病中起关键作用。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驱动。本申请的目的是鉴定调节内皮细胞胞吐作用的新型细胞因子。 囊泡运输是细胞生物学的一个基本重要过程。在胞吐过程中，囊泡将蛋白质从高尔基体运送到细胞外部。几个家族的蛋白质已被确定为介导胞吐，但我们的知识是零碎和不完整的胞吐机制。我们不知道：（1）细胞如何调节运输囊泡的数量，（2）出芽囊泡如何与供体隔室分离，或（3）囊泡如何靶向其正确的目的地。在胞吐途径中鉴定新的蛋白质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的实验模型。我们大部分关于运输的知识都依赖于对分泌神经囊泡的神经元的研究。然而，神经囊泡的胞吐是具有挑战性的研究：神经递质的测定既不快速也不稳健。相反，内皮细胞分泌血管性血友病因子到培养基中，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。为了验证这一假设，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：鉴定新的蛋白质，其通过调节内皮细胞的胞吐作用来调节内皮细胞的胞吐作用。 功能基因组筛选我们将进行大规模的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细胞胞吐作用的单个基因。 目的#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，我们将描述一个选定的几个候选基因产品确定 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发生、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先前在全基因组关联研究中确定的与人类内皮细胞胞吐作用改变相关的5个候选基因。 这一提议的意义在于囊泡运输途径是重要的，但尚未得到很好的理解，它们对人类血管炎性疾病如动脉粥样硬化至关重要。 本研究的创新之处在于：（1）鉴定了调控囊泡运输的新基因;（2）鉴定了新基因， 最近在人类中鉴定出的与胞吐作用相关的产物，（3）鉴定人类细胞中胞吐蛋白的新方法。

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.