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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

基本信息

批准号：
9975873
负责人：
MICHAEL S BOYCE
金额：
$ 32.21万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-09-21 至 2021-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9975873
关键词：
Animal Model Biochemical Biological Biological Assay Biology CRISPR/Cas technology Capsid 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

项目摘要

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.

三分之一的真核蛋白通过分泌途径靶向特定位置，