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: 10367509
• 负责人: MICHAEL S BOYCE
• 金额: $ 34.59万
• 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/10367509
• 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; Light; 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 - Cell Movement; 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.

项目总结/摘要 三分之一的真核蛋白质通过分泌途径分选到特定位置，包括 内质网（ER）、高尔基体、质膜或细胞外环境。因为错误定向的蛋白质 分泌途径对于建立和维持正常的细胞和组织至关重要 physiology. COPII外壳蛋白复合物，介导从ER的顺行运输，是一个关键的控制 蛋白质靶向点。事实上，COPII基因的突变会导致一系列人类疾病，包括颅- 骨缝发育不良（CLSD），骨生成障碍（OI）的一种亚型，血液系统疾病和各种 神经缺陷要了解COPII在细胞生理学中的作用，需要详细了解COPII的运输 并治疗其被破坏的疾病。然而，尽管COPII的核心机制定义明确， 已知脊椎动物细胞如何调节COPII活性以响应正常或病理信号或压力。 我们和其他人已经发现几种COPII蛋白被O-连接的b-N-乙酰葡糖胺修饰， （O-GlcNAc），细胞内蛋白质糖基化的动态形式。在上一个项目期开始时， O-GlcNAc酰化对COPII的影响仍然几乎完全未知。从那时起，我们确定了范围 核心COPII系统中O-GlcNAc酰化的研究，确定了囊泡中O-GlcNAc循环的功能效应 运输，设计了新的定量糖蛋白质组学方法来分析O-GlcNAc的变化， 分泌途径应激和其他刺激，并证明了特定的Sec 23 A O-GlcNAc位点是 所需的内源性胶原蛋白运输在培养的人细胞和软骨细胞的发展 斑马鱼总之，这些结果表明，COPII蛋白的位点特异性O-GlcNAc基化控制了 脊椎动物细胞和组织中的货物运输。然而，仍有一些重大问题没有得到解答，包括 O-GlcNAc对COPII蛋白的作用机制，COPII蛋白是调节COPII的上游刺激物。 早期分泌途径中的GlcNAc酰化和O-GlcNAc信号传导的全局景观。在这里，我们建议 在下一个颁奖期间解决这些重要问题。 在目的1中，我们将剖析O-GlcNAc循环影响COPII囊泡的分子机制 贩卖人口在目标2中，我们将鉴定控制COPII蛋白O-GlcNAc酰化的上游刺激物，并确定 这种信号在蛋白质分泌和细胞周期进程中的下游作用。在目标3中，我们将使用 糖蛋白质组学方法来调查响应COPII货物运输的蛋白质组范围的O-GlcNAc信号传导， 并提供蛋白分泌中O-GlcNAc酰化和磷酸化之间串扰的综合图像。 我们的工作将揭示O-GlcNAc如何调节细胞和组织的运输，并可能揭示新的 通过操纵蛋白质糖基化治疗COPII功能障碍疾病的机会。