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: 9384237
• 负责人: MICHAEL S BOYCE
• 金额: $ 32.58万
• 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/9384237
• 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.

所有真核蛋白的三分之一通过分泌途径以靶向特定位置， 包括内质网（ER），高尔基体，质膜或细胞外环境。自误导以来 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，介导从ER到高尔基体的囊泡运输的COPII蛋白复合物， 是蛋白质靶向的关键控制点。此外，COPII基因的突变引起了一系列人类 疾病，包括颅骨透明质酸核心发育不良（CLSD）和成骨的疾病（OI）。详细的 需要了解Copii囊泡运输才能了解其在细胞生理中的作用并治疗疾病 它被破坏了。但是，尽管核心COPII机械的定义很好，但对如何如何了解 哺乳动物细胞根据发育，代谢或病理提示调节COPII活性。 最近，我们和其他人发现几种COPII蛋白是通过O连接的B-N-修饰的 乙酰葡萄糖（O-GLCNAC），一种动态形式的细胞内蛋白糖基化。有趣的是，糖基化 COPII组件包括Sec23a和Sec24D，分别在CLSD和OI中突变，表现出来 在胶原蛋白失误和骨骼畸形中。但是，o-glcnacylation对copii的影响 途径仍然不清楚。在初步工作中，我们使用了一种化学生物学方法来表明至少四个 包括SEC23和SEC24在内的COPII成分，在O-GLCNAC介导的蛋白质 - 蛋白质相互作用中进行 人类细胞。此外，我们证明了O-GlCNAC循环的药理抑制作用阻碍了COPII 贩运。最后，我们发现Sec23a的不糖基突变体未能营救胶原蛋白贩运 Sec23a突变碎屑斑马鱼的骨骼生成缺陷。这些结果在一起表明站点 - 单个COPII蛋白的特异性O-Glcnacylation控制脊椎动物细胞和组织中的囊泡运输。 该项目的目的是定义O-Glcnacylation对机械和功能效应对 Copii途径。我们将通过三个具体目标来实现我们的目标。在AIM 1中，我们将剖析 O-GlCNAC循环对复印囊泡运输的功能影响。在AIM 2中，我们将定义特定地点的作用 人类细胞中SEC23A和SEC24D的O-Glcnacylation。在AIM 3中，我们将确定COPII的贡献 CLSD和OI的脊椎动物模型中的O-Glcnacylation。我们的工作将为O-Glcnacylation如何开发 调谐细胞和组织中的蛋白质运输，并可能揭示出治疗COPII疾病的新机会 功能障碍，例如CLSD和OI，通过靶向蛋白质糖基化。