Control of COPII vesicle trafficking by intracellular protein glycosylation

通过细胞内蛋白质糖基化控制 COPII 囊泡运输

• 批准号: 10541246
• 负责人: MICHAEL S BOYCE
• 金额: $ 33.02万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-21 至 2025-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10541246
• 关键词: Address; Award; Biochemical; Biological; Biological Process; Brefeldin A; Capsid Proteins; Cell Cycle Progression; Cell Proliferation; Cell membrane; Cell physiology; Cells; Cellular biology; Chemicals; Chondrocytes; Collagen; Complex; Cues; Data; Defect; Development; Disease; Dysplasia; Endoplasmic Reticulum; First Independent Research Support and Transition Awards; Functional disorder; Genes; Glucose; Goals; Golgi Apparatus; Hematological Disease; Hematology; Homeostasis; Human; Human Pathology; Individual; Inherited; Kinetics; Knowledge; Link; Location; Mammalian Cell; Mass Spectrum Analysis; Mediating; Membrane; Metabolic; Metabolism; Methods; Modification; Molecular; Mutation; Neurologic; Normal Cell; Normal tissue morphology; Nutrient; O-GlcNAc transferase; Osteogenesis Imperfecta; Pathologic; Pathway interactions; Phosphorylation; Physiological; Physiology; Prevalence Study; Protein Glycosylation; Protein Secretion; Protein Sortings; Proteins; Proteome; Proteomics; Regulation; Role; Signal Transduction; Sirolimus; Site; Skeleton; Sorting; Starvation; Stimulus; Stress; Surgical sutures; System; Tissues; Vertebrates; Vesicle; Work; Zebrafish; experimental study; extracellular; genetic approach; glycoproteomics; glycosylation; human disease; insight; interdisciplinary approach; nervous system disorder; novel; paralogous gene; peptide O-linked N-acetylglucosamine-beta-N-acetylglucosaminidase; pointed protein; protein complex; protein protein interaction; protein transport; response; sugar; trafficking

Project Summary/Abstract One third of eukaryotic proteins transit the secretory pathway for sorting to specific locations, including the endoplasmic reticulum (ER), Golgi, plasma membrane or extracellular milieu. Since misdirected proteins cannot function, the secretory pathway is critical for establishing and maintaining normal cell and tissue physiology. The COPII coat protein complex, which mediates anterograde trafficking from the ER, is a key control point for protein targeting. Indeed, mutations in COPII genes cause a range of human diseases, including cranio- lenticulo-sutural dysplasia (CLSD), a subtype of osteogenesis imperfecta (OI), hematologic disorders and myriad neurological defects. Detailed knowledge of COPII trafficking is required to understand its role in cell physiology and to treat disorders in which it is disrupted. However, while the core COPII machinery is well defined, little is known about how vertebrate cells regulate COPII activity in response to normal or pathological signals or stress. We and others have found that several COPII proteins are modified by O-linked b-N-acetylglucosamine (O-GlcNAc), a dynamic form of intracellular protein glycosylation. At the start of the prior project period, the effects of O-GlcNAcylation on COPII remained almost entirely unknown. Since then, we have defined the scope of O-GlcNAcylation in the core COPII system, identified functional effects of O-GlcNAc cycling in vesicle trafficking, devised new quantitative glycoproteomics methods to profile O-GlcNAc changes in response to secretory pathway stress and other stimuli, and demonstrated that particular Sec23A O-GlcNAc sites are required for endogenous collagen trafficking in cultured human cells and in the chondrocytes of developing zebrafish. Together, these results demonstrate that site-specific O-GlcNAcylation of COPII proteins governs cargo trafficking in vertebrate cells and tissues. However, major unanswered questions remain, including the mechanistic effects that O-GlcNAc exerts on COPII proteins, the upstream stimuli that modulate COPII O- GlcNAcylation and the global landscape of O-GlcNAc signaling in the early secretory pathway. Here, we propose to address these important questions in the next award period. In Aim 1, we will dissect the molecular mechanisms by which O-GlcNAc cycling influences COPII vesicle trafficking. In Aim 2, we will identify upstream stimuli that control COPII protein O-GlcNAcylation and determine the downstream effects of this signaling in protein secretion and cell cycle progression. In Aim 3, we will use our glycoproteomics methods to canvass proteome-wide O-GlcNAc signaling in response to COPII cargo trafficking, and provide an integrated picture of crosstalk between O-GlcNAcylation and phosphorylation in protein secretion. Our work will shed new light on how O-GlcNAc regulates trafficking in cells and tissues, and may reveal new opportunities to treat diseases of COPII dysfunction by manipulating protein glycosylation.

项目摘要/摘要 三分之一的真核蛋白传输了分泌途径，用于分类到特定位置，包括 内质网（ER），高尔基体，质膜或细胞外环境。由于蛋白质误导 无法发挥作用，分泌途径对于建立和维持正常细胞和组织至关重要 生理。 COPII外套蛋白复合物介导了从ER介导的顺行运输，是一个钥匙控制 蛋白质靶向的点。实际上，COPII基因中的突变会导致一系列人类疾病，包括Cranio- 胸膜核定性发育不良（CLSD），成骨的亚型（OI），血液学疾病和无数 神经缺陷。需要对COPII贩运的详细知识才能了解其在细胞生理中的作用 并治疗破坏的疾病。但是，虽然核心COPII机械的定义很好，但几乎没有 知道脊椎动物细胞如何根据正常或病理信号或应力调节COPII活性。 我们和其他人发现，几种COPII蛋白是通过O连接的B-N-乙酰葡萄糖修饰的 （O-GLCNAC），一种动态形式的细胞内蛋白糖基化。在上一期开始时， O-Glcnacylation对CoPII的影响几乎完全未知。从那以后，我们定义了范围 O-Glcnacylation在核心COPII系统中，囊泡中O-GlCNAC循环的功能效应 贩运，设计了新的定量糖蛋白质组学方法来介绍O-GLCNAC的变化，以响应于 分泌途径应力和其他刺激，并证明了特定的Sec23a O-GlCNAC位点是 内源性胶原蛋白运输在培养的人类细胞和发育中的软骨细胞中所必需的 斑马鱼。总之，这些结果表明，COPII蛋白的特定位点O-Glcnacylation控制 在脊椎动物细胞和组织中运输货物。但是，仍然存在重大未解决的问题，包括 O-GlCNAC对COPII蛋白发挥作用的机械作用，Copii蛋白是调节COPII O-的上游刺激 早期分泌途径中O-GLCNAC信号传导的Glcnacylation和全球景观。在这里，我们建议 在下一个奖励期间解决这些重要问题。 在AIM 1中，我们将剖析O-GlCNAC循环影响Copii囊泡的分子机制 贩运。在AIM 2中，我们将确定控制COPII蛋白O-Glcnacylation的上游刺激并确定 该信号在蛋白质分泌和细胞周期进程中的下游效应。在AIM 3中，我们将使用我们的 针对Copii货物贩运的糖蛋白质组学方法来绘制全蛋白质组的O-GLCNAC信号传导， 并提供蛋白质分泌中O-Glcnacylation和磷酸化之间的串扰的综合图片。 我们的工作将为O-GlcNAC如何调节细胞和组织中的运输方式提供新的启示，并可能揭示新的 通过操纵蛋白质糖基化治疗COPII功能障碍的疾病的机会。