Spatio-temporal regulation of ARF signaling in vesicle formation

ARF信号在囊泡形成中的时空调节

• 批准号: 9762124
• 负责人: ELIZABETH S SZTUL
• 金额: $ 33.73万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-05 至 2021-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9762124
• 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; 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; Vesicle Transport Pathway; anti-cancer; base; cell growth; cell motility; design; fitness; imaging approach; in vitro Assay; member; migration; neuroblastoma cell; novel; prevent; recruit; response; spatiotemporal; trafficking

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.

项目总结/摘要 双向囊泡运输是真核细胞将蛋白质运送到分泌细胞器的重要途径 和细胞表面，并从细胞中释放。据估计，30%的哺乳动物蛋白质组 必须通过分泌途径GBF 1（Golgi localized Brefeldin A-sensitive Factor 1）是一种关键的 GBF 1是从高尔基体到内质网逆行交通的调节因子，而GBF 1的活性是从高尔基体到内质网逆行交通所必需的。 分泌途径的建立和维持。GBF 1属于大鸟嘌呤家族 核苷酸交换因子（GEFs），是一种促进ARF上GDP/GTP交换的酶 小Ras样GTP酶亚家族。GBF 1介导的ARF激活是形成 逆行COPI囊泡，GBF 1代表COPI囊泡形成的上游调节因子，因为它 通过限制ARF激活来决定囊泡形成的时间和位点。然而，尽管批评 尽管GBF 1在细胞内稳态中的重要性，但我们仍然不知道GBF 1本身在细胞中是如何调节的。 具体来说，我们不知道细胞如何向GBF 1发出信号，以“通知”它细胞需要逆行交通 以及什么机制确保GBF 1仅在正确的时间启动COPI囊泡的形成， 对地方了本提案旨在阐明这一谜团。我们将检验细胞具有 抑制GBF 1活性以防止假性ARF激活的机制，但在一个特定的时间内释放这种抑制作用。 以时间和地点受限的方式响应指示逆行交通的信号通路 需求我们的目标是确定细胞如何翻译它们对ARF激活和膜转运的需要 空间和时间受限的GBF 1功能。我们提出了3个具体目标，以确定这些过程， 在所有细胞中调节GBF 1功能的信号通路，逆行COPI 这对分泌途径的稳态至关重要。在目标1中，我们将确定 通过识别细胞内的内在靶向信息， GBF 1和标记用于GBF 1募集的膜位点的膜组分。在目标2中，我们将 通过评估以下因素的作用，确定调节GBF 1在膜上催化活性的机制： 磷脂酰肌醇磷酸（PIP）在调节GBF 1催化活性中的作用。在目标3中，我们将确定 通过定义角色，协调GBF 1功能与COPI流量需求的信令路径 KDEL-R激活和PKA和SFK途径对GBF 1膜缔合和催化的影响 活动GBF 1广泛表达，对细胞和生物体健康至关重要; 从培养细胞中耗尽导致死亡，并且小鼠或果蝇敲除是胚胎致死的。 GBF 1在病理学背景中也很重要，因为它对于胶质母细胞瘤细胞的迁移是必需的 和复制人致病性肠道病毒。我们的研究将提供关键的新知识， GBF 1在基础细胞生理学中的调节，并将为治疗性药物的设计提供策略 干预以控制病理背景下GBF 1介导的事件。