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.
Our project focuses on COPII, a set of proteins that transports cargoes from the endoplasmic reticulum to the Golgi. COPII is required for healthy cell and tissue physiology, and mutations in COPII genes cause a variety of inherited human diseases, yet the mechanisms that cells use to regulate COPII activity are largely unknown. We will take an interdisciplinary approach to study a new form of COPII regulation in which sugars are attached to COPII proteins to adjust their functions.
| Chen, Po-Han; Chi, Jen-Tsan; Boyce, Michael (2018) Functional crosstalk among oxidative stress and O-GlcNAc signaling pathways. Glycobiology 28:556-564 |
| Tarbet, Heather J; Toleman, Clifford A; Boyce, Michael (2018) A Sweet Embrace: Control of Protein-Protein Interactions by O-Linked ?-N-Acetylglucosamine. Biochemistry 57:13-21 |
| Cox, Nathan J; Luo, Peter M; Smith, Timothy J et al. (2018) A Novel Glycoproteomics Workflow Reveals Dynamic O-GlcNAcylation of COP?1 as a Candidate Regulator of Protein Trafficking. Front Endocrinol (Lausanne) 9:606 |
| Cox, Nathan J; Unlu, Gokhan; Bisnett, Brittany J et al. (2018) Dynamic Glycosylation Governs the Vertebrate COPII Protein Trafficking Pathway. Biochemistry 57:91-107 |
