Spatio-temporal regulation of ARF signaling in vesicle formation

ARF信号在囊泡形成中的时空调节

基本信息

批准号：
9982347
负责人：
ELIZABETH S SZTUL
金额：
$ 29.14万
依托单位：
UNIVERSITY OF ALABAMA AT BIRMINGHAM
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-09-05 至 2022-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9982347
关键词：
Antiviral Agents Binding Biological Biotin Brefeldin A Cause of Death Cell physiology Cell surface Cells Coat Protein Complex I Cultured Cells Cyclic AMP-Dependent Protein Kinases Cytosol Drosophila genus Embryo Ensure Enterovirus Enzymes Estrogen receptor positive Eukaryotic Cell Event Family Family Picornaviridae Foundations GBF1 gene Glioblastoma Goals Golgi Apparatus Guanine Nucleotide Exchange Factors Guanosine Triphosphate Guanosine Triphosphate Phosphohydrolases Health Homeostasis Human Impairment In Situ KDEL receptor Knock-out Knowledge Label Life Lipids Maintenance Mediating Membrane Membrane Protein Traffic Molecular Monomeric GTP-Binding Proteins Mus Organelles PH Domain Pathogenicity Pathologic Pathway interactions Phosphatidylinositol Phosphates Phosphorylation Process Proteins Proteome Recruitment Activity Regulation Retrieval Role Route Signal Pathway Signal Transduction Site Specificity Testing Therapeutic Intervention Time Translating Transmembrane Transport Vesicle anti-cancer base cell growth cell motility design fitness imaging approach in vitro Assay member migration neuroblastoma cell novel prevent recruit response spatiotemporal trafficking vesicle transport

项目摘要

Project Summary/Abstract Bi-directional vesicular transport is vital in all eukaryotic cells to deliver proteins to secretory organelles and the cell surface, and for release from the cell. It is estimated that 30% of the mammalian proteomes must traffic the secretory pathway. GBF1 (Golgi localized Brefeldin A-sensitive Factor1) is a key regulator of retrograde traffic from the Golgi to the ER, and GBF1 activity is required for the establishment and maintenance of the secretory pathway. GBF1 belongs to a family of large Guanine nucleotide Exchange Factors (GEFs) and is an enzyme that facilitates GDP/GTP exchange on the ARF subfamily of small Ras-like GTPases. GBF1-mediated ARF activation is required for the formation of retrograde COPI vesicles, and GBF1 represents an upstream regulator of COPI vesicle formation as it dictates the time and site of vesicle formation by restricting ARF activation. Yet, despite the critical importance of GBF1 in cellular homeostasis, we remain ignorant of how GBF1 itself is regulated in cells. Specifically, we do not know how cells signal to GBF1 to “notify” it of a cellular need for retrograde traffic and what mechanisms ensure that GBF1 initiates COPI vesicle formation only at the right time and the right place. This proposal aims to illuminate this enigma. We will test the hypothesis that cells have mechanisms to inhibit GBF1 activity to prevent spurious ARF activation, but release such inhibition in a time and site-restricted manner in response to a signaling pathway that indicates retrograde traffic demand. Our goal is to define how cells translate their need for ARF activation and membrane transport into space- and time-restricted GBF1 function. We propose 3 specific aims to identify the processes and signaling pathways that regulate GBF1 function in an essential traffic routing in all cells, retrograde COPI traffic that is vital for the homeostasis of the secretory pathway. In Aim 1, we will identify the mechanisms that selectively target GBF1 to Golgi membranes by identifying the intrinsic targeting information within GBF1 and the membrane components that mark membrane sites for GBF1 recruitment. In Aim 2, we will define the mechanisms that regulate GBF1 catalytic activity at the membrane by assessing the role of phosphatidylinositol phosphates (PIPs) in regulating GBF1 catalytic activity. In Aim 3, we will determine the signaling pathways that coordinate GBF1 function with the need for COPI traffic by defining the role of KDEL-R activation and the PKA and SFK pathways on GBF1 membrane association and catalytic activity. GBF1 is ubiquitously expressed and critically important to cell and organismal health; GBF1 depletion from cultured cells causes death and a mouse or Drosophila knockout is embryonic lethal. GBF1 is also important in pathological contexts since it is essential for migration of glioblastoma cells and for replication of human pathogenic enteroviruses. Our studies will provide critical new knowledge of GBF1 regulation in basic cellular physiology and will inform strategies for the design of therapeutic intervention to control GBF1-mediated events in pathological contexts.

项目概要/摘要双向囊泡运输对于所有真核细胞将蛋白质递送至分泌细胞器至关重要据估计，30% 的哺乳动物蛋白质组是从细胞中释放出来的。必须通过分泌途径 GBF1（高尔基本地化 Brefeldin A 敏感因子 1）是关键。从高尔基体到内质网逆行交通的调节器，GBF1 活性是 GBF1 属于大鸟嘌呤家族的建立和维持。核苷酸交换因子 (GEF) 是一种促进 ARF 上 GDP/GTP 交换的酶 GBF1 介导的 ARF 激活是小 Ras 样 GTP 酶亚家族的形成所必需的。 GBF1 代表 COPI 囊泡形成的上游调节因子，因为它尽管至关重要，但通过限制 ARF 激活来决定囊泡形成的时间和部位。虽然 GBF1 在细胞稳态中的重要性，但我们仍然不知道 GBF1 本身如何在细胞中受到调节。具体来说，我们不知道细胞如何向 GBF1 发送信号以“通知”它细胞需要逆行流量以及什么机制确保 GBF1 仅在正确的时间启动 COPI 囊泡形成以及这个提议旨在阐明这个谜团，我们将检验细胞具有的假设。抑制 GBF1 活性以防止虚假 ARF 激活的机制，但在响应指示逆行交通的信号通路的时间和地点限制方式我们的目标是确定细胞如何转化其对 ARF 激活和膜运输的需求。我们提出了 3 个具体目标来识别过程和时间限制。在所有细胞的重要交通路由中调节 GBF1 功能的信号通路，逆行 COPI 对于分泌途径的稳态至关重要的交通在目标 1 中，我们将确定其机制。通过识别内部的内在靶向信息，选择性地将 GBF1 靶向高尔基膜 GBF1 和标记 GBF1 募集膜位点的膜成分在目标 2 中，我们将。通过评估 GBF1 催化活性的作用来定义调节膜上 GBF1 催化活性的机制在目标 3 中，我们将确定磷脂酰肌醇磷酸酯 (PIP) 调节 GBF1 催化活性的作用。通过定义角色来协调 GBF1 功能与 COPI 流量需求的信号传导通路 KDEL-R 激活以及 PKA 和 SFK 途径对 GBF1 膜缔合和催化的影响 GBF1 普遍表达，对细胞和生物健康至关重要；培养细胞的耗尽会导致死亡，而小鼠或果蝇基因敲除则导致胚胎致死。 GBF1 在病理情况下也很重要，因为它对于胶质母细胞瘤细胞的迁移至关重要我们的研究将为人类致病性肠道病毒的复制提供重要的新知识。 GBF1 在基础细胞生理学中的调节将为设计治疗策略提供信息干预以控制病理环境中 GBF1 介导的事件。