Novel Regulators of Endothelial Exocytosis

描述(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。血管炎症由几个不同的阶段组成，第一个阶段由内皮细胞胞吐作用驱动。这项应用的目标是确定调节内皮细胞胞吐的新细胞因子。囊泡运输是细胞生物学中一个至关重要的过程。在胞吐过程中，囊泡将蛋白质从高尔基体输送到细胞外部。有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碎的和不完整的。我们不知道：(1)细胞如何调节运输小泡的数量，(2)萌发中的小泡如何与供体隔室分离，或(3)小泡如何被定位到正确的目的地。识别胞外途径中的新蛋白质可能有助于回答这些基本问题中的一些。在这一领域取得进展的一个关键障碍在于历史实验模型。我们关于运输的大部分知识都依赖于对神经细胞分泌神经泡的研究。然而，神经囊泡的胞吐作用的研究是具有挑战性的：神经递质的分析既不快速也不可靠。相比之下，内皮细胞将von Willebrand因子分泌到介质中，这是一种易于高通量测量的蛋白质。我们假设，一组离散的蛋白质调节内皮细胞颗粒的胞吐。为了验证这一假设，我们计划在以下特定目标中识别和表征参与胞吐作用的蛋白质：目标1：识别通过一种新的蛋白调节内皮细胞胞吐作用。 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的蛋白质和途径。排除影响细胞活性的基因后，我们将验证增加或减少内皮细胞胞吐的单个基因。目的#2：描述调控内皮细胞胞吐作用的新蛋白的作用。接下来，我们将对已鉴定的几种候选基因产物进行鉴定。 在目标1中，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5个候选基因，它们与人类内皮细胞胞吐改变有关。这一建议的意义在于，囊泡运输途径很重要，但还没有被很好地理解，而且它们对人类血管炎症性疾病如动脉粥样硬化至关重要。这一建议的创新之处在于：(1)识别调控囊泡运输的新基因；(2)鉴定新基因 最近在人类中发现的与胞吐作用相关的产品，(3)鉴定人类细胞中胞外蛋白的新方法。

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.