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: 9750747
• 负责人: MICHAEL S BOYCE
• 金额: $ 30.86万
• 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 2021-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9750747
• 关键词: Animal Model; Biochemical; Biological; Biological Assay; Biology; CRISPR/Cas technology; Capsid Proteins; Carrier Proteins; Cell Line; Cell membrane; Cell physiology; Cells; Chemicals; Chondrocytes; Client; Collagen; Craniofacial Abnormalities; Cues; Cultured Cells; Data; Defect; Development; Disease; Dysmorphology; Dysplasia; Endoplasmic Reticulum; Epithelial Cells; Exhibits; Functional disorder; Genes; Goals; Golgi Apparatus; Growth Factor; Human; Human Pathology; Individual; Inherited; Knowledge; Light; Link; Location; Mammalian Cell; Maps; Mass Spectrum Analysis; Mediating; Metabolic; Methods; Modeling; Mutate; Mutation; Normal Cell; Normal tissue morphology; Osteogenesis Imperfecta; Pathologic; Pathway interactions; Pharmacology; Physiological; Physiology; Pilot Projects; Play; Protein Glycosylation; Protein Sortings; Proteins; Proteomics; Regulation; Role; Set protein; Signal Transduction; Site; Stress; Surgical sutures; Testing; Tissues; Vertebrates; Vesicle; Work; Zebrafish; extracellular; genetic approach; genome editing; glycoproteomics; glycosylation; human disease; insight; interdisciplinary approach; intracellular protein transport; live cell imaging; mutant; novel; overexpression; pointed protein; protein complex; protein protein interaction; protein transport; response; skeletal; skeletogenesis; sugar; trafficking

One third of all eukaryotic proteins pass through the secretory pathway for targeting 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. In particular, the COPII protein complex, which mediates vesicle trafficking from the ER to the Golgi, is a key control point for protein targeting. Moreover, mutations in COPII genes cause a range of human diseases, including cranio-lenticulo-sutural dysplasia (CLSD) and osteogenesis imperfecta (OI). Detailed knowledge of COPII vesicle 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 mammalian cells regulate COPII activity in response to developmental, metabolic or pathological cues. Recently, we and others found that several COPII proteins are modified by O-linked b-N- acetylglucosamine (O-GlcNAc), a dynamic form of intracellular protein glycosylation. Interestingly, glycosylated COPII components include Sec23A and Sec24D, which are mutated in CLSD and OI, respectively, manifesting in collagen mistrafficking and skeletal dysmorphology. However, the effects of O-GlcNAcylation on the COPII pathway remain unclear. In preliminary work, we used a chemical biology approach to show that at least four COPII components, including Sec23 and Sec24, engage in O-GlcNAc-mediated protein-protein interactions in human cells. In addition, we showed that pharmacological inhibition of O-GlcNAc cycling hinders COPII trafficking. Finally, we found that an unglycosylatable mutant of Sec23A failed to rescue the collagen trafficking and skeletogenesis defects of Sec23A-mutant crusher zebrafish. Together, these results suggest that site- specific O-GlcNAcylation of individual COPII proteins governs vesicle trafficking in vertebrate cells and tissues. The objective of this project is to define the mechanistic and functional effects of O-GlcNAcylation on the COPII pathway. We will accomplish our objective through three Specific Aims. In Aim 1, we will dissect the functional impact of O-GlcNAc cycling on COPII vesicle trafficking. In Aim 2, we will define the role of site-specific O-GlcNAcylation of Sec23A and Sec24D in human cells. In Aim 3, we will determine the contribution of COPII O-GlcNAcylation in vertebrate models of CLSD and OI. Our work will shed new light on how O-GlcNAcylation tunes protein trafficking in cells and tissues, and may reveal new opportunities to treat diseases of COPII dysfunction, such as CLSD and OI, by targeting protein glycosylation.

所有真核蛋白质的三分之一通过分泌途径靶向特定位置， 包括内质网 (ER)、高尔基体、质膜或细胞外环境。既然方向错了 蛋白质无法发挥作用，分泌途径对于建立和维持正常细胞和组织至关重要 生理。特别是 COPII 蛋白复合物，它介导从内质网到高尔基体的囊泡运输， 是蛋白质靶向的关键控制点。此外，COPII 基因突变会导致一系列人类疾病 疾病，包括颅骨豆状缝发育不良（CLSD）和成骨不全症（OI）。详细的 需要了解 COPII 囊泡运输的知识才能了解其在细胞生理学中的作用并治疗疾病 它被扰乱了。然而，虽然 COPII 的核心机制已经明确定义，但人们对其如何运作却知之甚少。 哺乳动物细胞根据发育、代谢或病理线索调节 COPII 活性。 最近，我们和其他人发现一些 COPII 蛋白被 O-连接的 b-N- 修饰。 乙酰氨基葡萄糖 (O-GlcNAc)，细胞内蛋白质糖基化的动态形式。有趣的是，糖基化 COPII组件包括Sec23A和Sec24D，它们分别在CLSD和OI中发生突变，表现为 胶原蛋白错误运输和骨骼畸形。然而，O-GlcNAc 酰化对 COPII 的影响 途径仍不清楚。在前期工作中，我们使用化学生物学方法表明至少有四种 COPII 成分，包括 Sec23 和 Sec24，参与 O-GlcNAc 介导的蛋白质-蛋白质相互作用 人体细胞。此外，我们发现 O-GlcNAc 循环的药理抑制会阻碍 COPII 贩运。最后，我们发现 Sec23A 的不可糖基化突变体未能挽救胶原蛋白运输 以及 Sec23A 突变体斑马鱼的骨骼形成缺陷。总之，这些结果表明站点- 单个 COPII 蛋白的特异性 O-GlcNAc 酰化控制脊椎动物细胞和组织中的囊泡运输。 该项目的目标是确定 O-GlcNAcylation 对 COPII途径。我们将通过三个具体目标来实现我们的目标。在目标 1 中，我们将剖析 O-GlcNAc 循环对 COPII 囊泡运输的功能影响。在目标 2 中，我们将定义特定站点的角色 人体细胞中 Sec23A 和 Sec24D 的 O-GlcNAc 酰化。在目标 3 中，我们将确定 COPII 的贡献 CLSD 和 OI 脊椎动物模型中的 O-GlcNAcylation。我们的工作将为 O-GlcNAcylation 提供新的线索 调节细胞和组织中的蛋白质运输，并可能揭示治疗 COPII 疾病的新机会 通过靶向蛋白质糖基化来治疗功能障碍，例如 CLSD 和 OI。